MLS 431 Digital Pathology & Photography PDF

1|Page MLS 431 LECTURE NOTE Learning objectives: At the end of this lecture, the students should be able to understand: 1. The concept of Digital Pathology and its key 8. The concepts of Photomicrography and aspects Ph...

1|Page MLS 431 LECTURE NOTE Learning objectives: At the end of this lecture, the students should be able to understand: 1. The concept of Digital Pathology and its key 8. The concepts of Photomicrography and aspects Photomacrography 2. Digital image analys (DIA): Applications, 9. Benefits of Digital Photography benefits and limitations 10. The general features of photomicrography 3. The concepts of Virtual Microscopy (VM) and 11. How to prepare stained sections for photography Virtual slides in pathology 12. How to evaluate a photomicrograph for 4. The components and workflow of VM publication 5. The applications, advantages and disadvantages 13. How to make excellent Photomicrographs with a of VM charge coupled device (CCD) camera 6. The importance of Photography in Morbid 14. How to prepare very low-power photographs of anatomy whole sections 7. Benefits of Digital Photography 15. How to label photomicrographs (Annotation) 16. Common faults in photomicrography  INTRODUCTION Previously, pathologists used to give opinion on holistic viewing of the image of the tissue/cells. Presently there is increasing demand to provide semi-quantitative features along with the quantitative information of various biomarkers on the tissue section or cytology smear. The massive development of the computer technology followed by marked advancement in the field of high-resolution image digitization, image storage, extraction of features and analysis. Digital pathology is a subfield of pathology that involves the use of digital technology to analyze and interpret pathological images. It involves the conversion of glass slides into digital images, which can then be analyzed using specialized software. Nowadays it is essential to have a good knowledge on digital image analysis (DIA) and virtual microscopy (VM) for the effective improvement of reporting tissue specimen.  KEY ASPECTS OF DIGITAL PATHOLOGY Some key aspects of digital pathology include: 1. Digital slide scanning: Converting glass slides into digital images. 2. Image analysis software: Analyzing and interpreting digital images. 3. Telepathology: Remote consultation and collaboration. 4. Artificial intelligence: Using AI and machine learning to analyze images. 5. Digital image storage and management: Storing and managing digital images.  2|Page  INTRODUCTION - Digital image analysis (DIA) refers to the use of computer algorithms and software to analyze and extract meaningful information from digital images of microscope slides, such as: o Cell counting and classification o Tissue segmentation and quantitation o Staining intensity and pattern analysis o Nucleus and cellular feature extraction o Detection of specific structures or features o Image segmentation o Machine learning-based classification - DIA provides more objective and consistent information of the images and also helps in the diagnosis, grading and classification of the disease. It provides various prognostic information and guidance to therapy. - Common techniques used in DIA include: o Machine learning o Image processing (filtering, thresholding and segmentation.) o Feature extraction and analysis o Pattern recognition and classification.  APPLICATIONS OF DIGITAL IMAGE ANALYSIS Education: The digital images are used for teaching. Telepathology: Digital slides are used for immediate online opinion. Detection of malignancy: Malignant cells are detected such as in cervical smear. Detailed morphologic study of DNA and chromosomes. Pattern recognition and grading of carcinoma. Quantitation of immunohistochemistry. Assessment of aggressiveness of a tumor for personalized medicine  BENEFITS OF DIGITALIMAGE ANALYSIS Same with benefits of digital pathology as stipulated in page 7  CHALLENGES AND LIMITATIONS Such as: 1. Image quality and standardization 2. Regulatory and legal issues 3. Integration with existing systems 4. Cybersecurity and data protection 3|Page  INTRODUCTION Training and education of morbid anatomy are based on the glass slides and traditional microscopy, text book and web pictures. However, there has been a massive advancement in digital technology in the last few years, whereby a complete digital slide is generated and available on a computer as ‘whole slide imaging’. Any part of the slide can be examined by the observer by zooming (increasing or decreasing the magnification) the particular area of the section immediately like the glass slide under the microscope. WHAT IS VIRTUAL MICROSCOPY? - Virtual microscopy refers to the digital scanning and viewing of glass slides, allowing for remote examination, diagnosis and consultation. - It involves: o Whole-slide imaging (WSI): Digitizing entire glass slides at high resolution o Digital slide analysis: Viewing and analyzing digital slides using specialized software o Telepathology: Sharing and consulting on digital slides with remote experts. WHAT ARE VIRTUAL SLIDES? - Virtual slides are digital images of glass microscope slides that have been created using digital slide scanning technology and can be viewed and analyzed on a computer or mobile device. - Virtual slides typically: o Are created using whole-slide imaging (WSI) scanners o Contain high-resolution images (often up to 40x magnification) o Preserve the original slide’s context and detail o Can be viewed using virtual microscopy (VM) software o Enable remote examination, diagnosis and consultation  COMPONENTS AND WORKFLOW OF VIRTUAL MICROSCOPY The components of VM include Digital Slide Scanner, Image Management software, Virtual Microscopy software and High-Speed Internet connection. The workflow is as follows: Slide scanning image acquisition image storage Virtual Microscopy software Remote access and viewing  APPLICATIONS OF VIRTUAL MICROSCOPY/SLIDES - Primary diagnosis: Virtual slide review for routine diagnosis - Second opinions, consultations, 4|Page - Education & training: Interactive learning, online courses and workshops. - Research and Collaboration: Shared access to digital slides, collaborative research - Digital pathology: Whole slide imaging, automated analysis - Immunohistochemistry: Digital slide analysis for IHC staining - Image analysis: Quantification, pattern recognition and machine learning - Frozen section diagnosis: Intraoperative consultations. - Digital slide review for tissue & cytological diagnosis, molecular testing, surgical specimens, autopsy cases. - Telepathology and remote consultation - Quality control & quality assurance.  ADVANTAGES OF VIRTUAL MICROSCOPY/SLIDES i) Multiple users can view the slides. ii) Only a computer and Internet access are enough to teach. iii) Same images are seen both by students and the teacher so better interaction is possible. iv) No need to have multiple microscopes and their maintenance. v) Consistent classical cases can be stored as VSs vi) More than one slide can be viewed in a same screen to compare the lesion. vii) The specific area or the particular object can be labelled for attention and display. viii) No fading or damage of digital slides. ix) No need of preparing multiple slides/sections from tissue block and thereby tissue is not exhausted. x) The cases can be accessed instantly and there is no need of museum staff. xi) Distant teaching (tele-education) and even self-learning. xii) Virtual workshop can be conducted efficiently. Role of Virtual Slides in Web-Based Teaching: The use of VM in web-based teaching provides the following benefits: - Enhances student learning experience - Facilitates interactive and immersive learning - Supports personalized and self-paced learning - Enables remote and flexible learning  DISADVANTAGES OF VIRTUAL MICROSCOPY/SLIDES i) Initial high cost of the equipments ii) Need of huge storage space iii) Unfamiliarity to handle the conventional. There will be limited tactile experience. iv) Image quality and resolution v) Technical issues and compatibility vi) Dependence on technology 5|Page PHOTOGRAPHY IN MORBID ANATOMY  INTRODUCTION In gross anatomy to microscopy (histology), pictures are data. The cost of space is insignificant compared to the expense of getting the experiment to the microscope. Therefore, it is utmost importance to take pictures. This material gives the student the background for making excellent photomicrographs and thus includes an expanded step- by-step procedure in making them. In general, photography is divided into two parts: - film cameras and film micrographs. - digital cameras and digital photomicrography Photographers control the camera and lens to ‘expose’ the light recording material (such as film) to the required amount of light to form a ‘latent image’ (on film) or ‘raw file’ (in digital camera) which, after appropriate processing, is converted to a usable image. Modern digital cameras replace film with an electronic image sensor based on light- sensitive electronic such as charge-coupled device (CCD) or complementary metal- oxide-semiconductor (CMOS) technology. The resulting digital image is stored electronically, but can be reproduced on paper or film.  PHOTOMICROGRAPHY This is the process of taking a photograph through a microscope or similar device to show a magnified image of an object. Photomicrographs can be imaged utilizing brightfield, phase contrast, and polarized light microscopy. The need of photography has increased enormously and now plays an essential part in Teaching, Medical research, Diagnostics and Scientific publications. It is essential then that all histologists and histoscientists to know how to prepare sections that are ideal for photography and also be aware of its principle. The use of photography to capture images in a microscope dates back to the invention of photographic process. Photographic techniques have been applied to recording images from the microscope for more than 170 years – an early example was Foucault’s micrograph of blood cells in 1844. Early photomicrographs were remarkable for their quality, but the techniques were laborious and burdened wilt long exposures and a difficult process for developing emulsion plates. The primary medium for photomicrography was film until the past decades when improvements in electronic camera and computer technology made digital imaging cheaper and easier to use than conventional photography. 6|Page Digital Photomicrography At the heart of a digital camera is the charge coupled device (CCD). A CCD is the film of digital photomicrograph. For the purpose of this lecture, we will consider the CCD as a light-collecting device composed of a specific number of discrete light collecting elements (pixels). When light strikes a pixel, the photons are converted to electrons that are stored in the pixel. The more electrons that are stored in the pixel, the brighter the pixel is. A CCD consists of a two-dimensional array of pixels. For the purposes of photomicrography, the important parameters of a CCD are: - The number of pixels, - The dynamic range (maximum number of electrons per pixel), - The signal to noise ratio, - The readout rate - The spectral sensitivity. OLYMPUS BX-60 MICROSCOPE DINO-LITE AM7025X WITH PM30 CAMERA EYEPIECE CAMERA AUTOMATIC CAMERA SYSTEM FITTED TO MICROSCOPE 7|Page  PHOTOMACROGRAPHY This is also known as Macrophotography. This is a type of close-up photography in which the image projected on a ‘film plane’ (film or digital electronic sensor) is as large or larger than the object or subject. E.g. if a photographer wants to take a macrophotograph of a specimen, he takes the camera to and fro until the object is in focus before he takes the snapshot. In Morbid anatomy, it is useful if, as the specimens are received, they are photographed, as the photographs will provide a permanent record and can be used in lectures, tape slide programmes, possible publication and as a service to donors of the specimens. Photography is best carried out when the specimen is in its fresh state, and after any initial dissection has taken place.  BENEFITS OF DIGITAL PHOTOGRAPHY There are several compelling reasons to convert to digital technology. The advantages/benefits of digital photography include: 1. The most persuasive is lower running costs and shorter image production turnaround times. Following the initial capital investment, additional costs are minimal. Increased efficiency and productivity is achieved for better teaching, education and patient care. 2. Memory cards for temporary storage of images are reusable and storage media such as CD-ROMs are inexpensive. 3. Results are almost instantaneous and images can be edited, and the quality and composition improved. 4. It is easier to catalogue, archive and disseminate/transmit a digital image than a print or 35-mm slide. This enhances collaboration & consultation, and facilitates research and education. 5. Multiple digital copies in different formats are possible. 6. New range of applications are possible such as telemicroscopy/ telepathology, image processing and analysis, digital slide archiving etc  GENERAL FEATURES OF PHOTOMICRORAPHY As described by Drury et.al. (1967), a few points that may affect all photomicrographic apparatus would include: Complete absence of all movement and vibration is essential because simple attachment cameras suffer from lack of this. A solid horizontal optical bench is needed for the microscope. Illumination is critical and must be centred, uniform and exactly reproducible. 8|Page Chromatic aberration must be minimized by the use of achromatic, fluorite and apochromatic objectives; the chromatic difference of magnification can be corrected by a compensating eyepiece. Photomicrography, like Histology calls for time and patience in order to obtain a standard technique.  PREPARATION OF STAINED SECTIONS FOR PHOTOGRAPHY The human eye can make allowances for slight defects in stained sections that may be suitable for student teaching or for diagnosis but the camera makes no such compromises and tends to emphasize technical imperfections. Hence to produce perfectly cut and stained sections suitable for microscopical examination for use with a micro-projector and for photomicrography, the following should be considered: - That the tissue must be free from cracks, since these will appear in the section. - It must be fresh, with no significant autolysis and be well fixed. - Fixation artifacts should be avoided. - Sections must be completed, with no creases, tears of scores. - Thin sections (5uuu or less) are essential for high power and oil immersion magnification. - If very low-power photomicrographs are required, sections of 10 – 15 uuuu thick should be cut (Thin sections are undesirable). - Staining must be clear and precise. - Filters are used to increase or diminish colours in black and white photographs. - Good contrast is needed for colour photography and for low-power monochrome. - Mounting of the sections must be on perfectly clean slides, with clean mountant and coverslips. - A large amount of adhesive should be avoided since this can produce background colouration. - Do not use a large amount of mounting medium.  HOW TO PREPARE COLOUR PHOTOMICROGRAPHS FOR PUBLICATION Histological Sections In general, 5 micrometer sections are desirable for photomicrography using the 1 Ox, 20x, and 40x objectives, and for the l00x objective with its depth of focus, a 3 micrometer section is preferable. For very low objectives such as the 4x, thicker sections (6-7 micrometers) may be necessary, if the tissue is not dense. Optimal H&E staining ensures the differential eosinophilia of the tissue components which is critical for best results.  9|Page With proper differentiation of the eosin, all components e.g. edema, collagen, keratin, and muscle, stain a different shade of pink or red. This differential staining occurs only with alcoholic solutions of eosin and if the eosin is differentiated in 95% ethanol (Luna, 1992). differential eosin staining is not a matter of personal preference, but is essential. Undifferentiated eosin-stained tissues lose too much clarity in the final photograph because all the tissues record the same red or pink. Phloxine which greatly reduces differential staining should not be added to the eosin. Hematoxylin should stain the nuclei blue-black as possible, without blocking out the detail of the chromatin. Harris’s hematoxylin is preferred as it produces a bluish-black, nuclear stain. Failure to properly differentiate the hematoxylin using acid-alcohol will result in inappropriate hematoxylin-staining of the cytoplasm (DMA, 1992). Microscopy - The microscope must be set up for Kohler illumination for each objective, and this should be checked when taking each photograph To set up Kohler illumination: Switch on the light source and make sure that light is coining through the field diaphragm at the base (upright microscope) or the top (inverted microscope) of the microscope stand. It may help to place a piece of paper over the field stop to see the light. Place your specimen on the stage and turn the nosepiece (which holds the objective lenses) to the 10X or 20X lens. Open the field diaphragm as far as it will go. Notice whether or not your specimen is illuminated. It will help to place a piece of paper over the top of the specimen to see light is getting through to it. If you are using the brightfield condenser stop, open the iris diaphragm (or aperture diaphragm) on the condenser turret (which contains the stops for brightfield and phase, etc) wide open to give the maximum illumination. If there is a swing-in front lens for the condenser (directly above (inverted) or below (upright) the specimen), you may need to swing it into the light path. The front lens should be about 1-3 mm above or below the specimen. There are condenser focusing knobs to do this. Now bring your specimen into focus with the coarse and fine focusing knobs. The best way to do this is to rack the lens as close as possible to the specimen watching the objective lens all the time (and NOT looking into the oculars) to make sure that the lens does not run into the slide. Then rack the lens away from the stage (or vice versa) while looking through the oculars to bring the specimen into focus (details are as sharp as they can be). If the light is too bright, reduce it with the rheostat on the light source. When the specimen is in focus, start to close the field diaphragm and also begin to carefully move the condenser up and down with the condenser focusing knobs. Look  10 | P a g e for a sharp image of the edge of the field diaphragm. This may be a little with a long working distance condenser. Also, f the iris diaphragm in the condenser turret is open wide, the glare may obscure the edge of the field diaphragm silhouette somewhat furthermore and you may find that this edge is not centered. When the edge of the field diaphragm silhouette is sharply defined, center it with the two knobs (usually knurled knobs) coming out diagonally from the condenser. Close down the field diaphragm most or all the way to get it centered properly. When it is centered, open the field diaphragm until its edge is outside the field. If you are doing brightfield or differential interference microscopy, do not yet open the field diaphragm. As stated before, you may notice some glare around the edge of the field diaphragm, that the edge area outside the edge is not completely dark like the outer part of the whole field as you should see it now. This glare comes from light bouncing around in the light path and going in and illuminating the specimen in such a way as to obscure detail in the specimen. To reduce this glare, close down the iris aperture in the condenser turret until all of the dark area outside of the field stop silhouette is evenly dark. Now open up the field diaphragm until the edge of the diaphragm silhouette is outside the field of view. You should also now be able to turn up the light at the power source. Your specimen should be properly illuminated and should give you a great image if it does not, check to make sure your lenses and other optical components are clean. Then recheck to see that you have followed each step properly. - Photomicrography should be performed in the morning when the eyes are minimally fatigued - Focus should be carefully adjusted for each photograph. - In general, the use of 10x or 20x objective provides the best contrast and visibility of the subject in most sections. The 4x objective should be used only when absolutely necessary to show the overall pattern, such as that of a neoplasm.  STEPS IN MAKING EXCELLENT PHOTOMICROGRAPHS WITH A CCD CAMERA A. Prepare the Microscope and Camera 1. Clean the lenses. 2. Set up Köhler illumination. 3. Adjust the transmitted illuminator’s voltage to 9 - 12 volts and turn on the epi- illumination light source to allow it to stabilize. 4. Remove all colored filters from the transmitted and epi-fluorescence light paths. 5. Set the camera system to the resolution at which you wish to make your photographs. 11 | P a g e 6. Image an empty field by transmitted light, turn off any gain or offset settings, adjust the camera’s exposure setting to give a neutral gray field, and perform a white balance. B. Prepare your Slides 1. Clean your slides on both sides. 2. Try to insure that only one cover glass is present. 3. Place the slide on the microscope stage with the specimen side facing the objective lens. C. Make Final Adjustments and Shoot 1. Adjust Köhler illumination for the objective lens in use. You can stop down the field iris to just outside the area that will be in the photograph; you should carefully adjust the condenser iris to achieve the best contrast without creating diffraction artifacts. 2. Focus: at magnifications below 10 X use a focus aid or magnifier if one is present. If using a high dry objective lens with a cover glass correction collar, adjust the collar for the sharpest possible image with the condenser iris fully open, then re- close the iris. If you are using an oil immersion lens, check for bubbles by observing the objective’s back focal plane. 3. Adjust the exposure time using neutral density filters and the camera’s exposure controls to achieve a relatively short exposure - between 1/10 and 1/100 second. In fluorescence microscopy the exposures will be longer so check for vibrations, focus drift and stage creep. 4. Recheck the focus by observing the image on your monitor, block the eyepieces if this is not done automatically, allow the microscope to settle, and capture the image. 5. Save the image as an uncompressed TIF image file.  HOW TO PREPARE VERY LOW-POWER PHOTOGRAPHS OF WHOLE SECTIONS Below is the method as described by Drury et. al., (1967) when photographic plate was in use in photographic processes. Method: 1. Take section to water 2. Stain with Weigert’s Iron Haematoxylin 3. Stain with a saturated aqueous solution of picric acid 4. Rinse in water, pass rapidly through alcohol to xylene and mount in synthetic resin medium 5. Place the section in photographic enlarger and project the image at desired magnification upon a non-yellow sensitive photographic plate. The plate is exposed and developed in the usual way. 12 | P a g e  HOW TO EVALUATE COLOUR PHOTOMICROGRAPHS FOR PUBLICATION Evaluating color digital photomicrographs for publication involves assessing technical quality, accuracy, and aesthetic appeal. Here's a comprehensive checklist: Technical Quality: 1. Gross defects: Defects such as lack of sharp focus or histological artifacts such as microtome knife marks or over thick sections should be evaluated first. Any photomicrograph containing the obvious defects should be rejected. 2. Resolution: Ensure the image has sufficiently high resolution (at least 300 dpi) for clear details. 3. Focus and Sharpness: Verify sharp focus, especially in critical areas. 4. Exposure: Check for proper exposure, avoiding over/under-exposure. 5. Color accuracy: Verify that the colors accuratelty represent the specimen or sample. Staining contrast and proper differentiation of stains should be assessed. For instance, in an ideal H&E staining, hematoxylin stained nuclei should be very dark (dark blue or even blue black colour). Eosin stained structures should be differentiated. In other words, all eosin stained tissues depicted in the photomicrograph (e.g collagen, smooth muscle, keratin, serum, blood cells and eosinophilic granules) should be stained different shades of red or pink. If the eosin is undifferentiated, these components will be as much the same colour and thus will not be easily identified in the photomicrograph. Such photomicrographs should be returned to the author with the advice to use correctly differentiated hematoxylin and eosin stained sections. - Thus to confirm accurate color representation, one should consider: a. Tissue staining (e.g., H&E, IHC)- b. Instrument calibration c. Color space (e.g., RGB, CMYK) The microscope’s clear background should have no colour. It should be extremely light grey. Colour casts are a common problem and are unacceptable. They can be removed by a computer programme such as Adobe Photoshop. 6. Noise and artifacts: Check for digital noise or artifacts (e.g., dust, scratches) which may distort the image. Ensure they are minimized. 7. Visibility of the lesion and suitability of the magnification too high or too low, inclusion of adjacent landmarks so that the viewer may orient himself/herself. 8. Composition and anatomically correct orientation: for e.g., the surface of the skin section should be horizontal with the surface on top. 9. Ethical considerations: Verify that the image has not been manipulated or falsified. 13 | P a g e Accuracy: 1. Representative image: Ensure the image accurately represents the histopathological feature. 2. Diagnostic relevance: Verify the image supports the research findings and diagnosis. 3. Labeling and annotation: Check for accurate labeling, arrows, and annotations. 4. Magnification and scale: Confirm correct magnification and scale bars. Aesthetic Appeal: 1. Composition: Balance and arrange elements for clear visualization. 2. Contrast: Optimize contrast for visual clarity. 3. Color harmony: Ensure color consistency and harmony. 4. Image cropping: Crop images to focus attention on relevant areas.  LABELLING OF PIIOTOMICROGRAPHS (ANNOTATION) One of the best practices to master is the use of Annotation on photomicrographs. Few decades ago, photomicrographs in prints which are meant for enhancing teaching value or for publication were labeled using letters (alphabets), numbers and symbols (e.g. arrows) superimposed on a paper sheet (known as Letracets). These annotations were used for labeling the specific points of interest on the photomicrographs. A key is usually placed beside or below the micrograph to explicate the illustrations. In recent times, annotation on digital images have become conventional and it involves highlighting features of interest to facilitate accurate diagnosis, education and research. Below is a step-by-step technique for annotating digital pathology images: 1. Image selection: Choose a high-quality image. 2. Annotation software: Utilize softwares or tools such as Adobe Photoshop or illustrator, Aperio ImageScope or ePathology software, Open-source tools like GIMP or Fiji etc. 3. Annotation types: a. Arrows: Indicate specific features or lesions b. Circles or Ellipses: Highlight tumour borders, nuclei, or cells c. Rectangles: Draw attention to specific regions d. Freehand drawing: Outline complex features or patterns 4. Color coding: Use distinct colours for different features or structures. 5. Labelling: Add text labels to explain annotations. Include relevant information (e.g. diagnosis, stain, magnification) 6. Measurement tools: Calibrate and use measurement tools for accurate sizing. Document measurements in the label or in reports. 14 | P a g e 7. Saving and Sharing: Save the annotated images in suitable formats (e.g. JPEG, TIFF). Share with colleagues or export for presentation/publications  COMMON FAULTS IN PHOTOMICROGRAPHY Photography through the microscope is undergoing a transition from film to digital imaging. New digital technologies are producing higher resolution micrographs, but the quality still falls short of that obtainable with film. Microscope configuration errors represent the greatest obstacle to quality photomicrographs, followed by errors in filter selection, film choice, aberration, dirt and debris, and processing mistakes. The most common fault is the use of unsatisfactory raw material, i.e. a poor quality section badly stained. However, the main technical problems are: a) Illumination ‘fall off’: this is particularly a problem in low-power photomicrography. It implies uneven illumination of the field which is an indication that the microscope has not been correctly set up for Kohier illumination. It is important to set the microscope up for each and every field. b) Yellow background: This occurs when an inappropriate colour temperature is used. It can be corrected by increasing the light intensity and the insertion of a suitable blue colour correction filter. c) Picture out of focus: This error is commonly caused by incorrect adjustment of the field image. Since each individual’s eyes are different it is necessary to adjust the individual eye piece to suit the photographer. Most cameras incorporate a special focusing grid; the operator must adjust individual eye pieces so that the grid is in sharp focus for each eye. d) Incorrect exposure: If an automatic exposure meter is used, check that DIN/ASA setting is appropriate to the film being used. This problem may be overcome by running test strips to find the best exposure.