Biophotonics and Bioimaging Overview

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Questions and Answers

What effect does using a linearly polarised window that matches the light source's polarisation have on diffuse light?

It filters out the diffuse light (correct)

It enhances the diffuse light

It has no effect on the diffuse light

It magnifies the diffuse light

In reflection imaging, what type of light needs to be discriminated against along with coherently back-scattered light?

Multiply back-scattered light (correct)

Directly transmitted light

Multipath reflected light

Ambient light

What is the primary purpose of time gating in optical imaging?

To change the light's polarisation

To magnify the captured image

To increase light intensity

To allow only specific photons to be detected (correct)

Which technique uses interference between a reference signal and back-scattered light for imaging?

Optical coherence tomography (OCT) (D) Signup and view all the answers

What method employs a confocal aperture for spatial filtering during reflection imaging?

Confocal imaging (D) Signup and view all the answers

What type of photons do not scatter and take the shortest route to the detector?

Ballistic photons (D) Signup and view all the answers

Which type of photon is categorized as slightly scattered but maintains some directionality?

Snake photons (D) Signup and view all the answers

What technique uses a confocal aperture to reject off-axis photons?

Spatial filtering (B) Signup and view all the answers

Which type of photons generally carries the least information?

Diffuse photons (B) Signup and view all the answers

In polarisation gating, why are snake photons partially depolarised?

They are illuminated with linearly polarised light. (C) Signup and view all the answers

Which characteristic of ballistic photons aids in their collection during imaging?

Maintaining the same polarisation (B) Signup and view all the answers

Why must diffuse photons usually be removed from measurements?

They are highly scattered and contain little information. (C) Signup and view all the answers

What is a significant advantage of employing spatial filtering during optical imaging?

It allows the collection of mainly ballistic and some snake photons. (B) Signup and view all the answers

What is a primary characteristic of fluorescence as an optical bioimaging technique?

It allows probing of biological structures both in vitro and in vivo. (A) Signup and view all the answers

What is the relationship between the energy of excitation and the energy of emission in fluorescence?

Energy of excitation is greater than energy of emission. (C) Signup and view all the answers

Which of the following statements is true regarding fluorophores in fluorescence microscopy?

Fluorophores bind with specific molecules for tagging purposes. (C) Signup and view all the answers

Which property makes fluorescence a suitable technique for analyzing small biological samples?

High signal-to-noise ratio. (A) Signup and view all the answers

Which of the following techniques is not categorized under optical imaging methods?

Nuclear Magnetic Resonance (C) Signup and view all the answers

What is one application of spatially resolved spectroscopy in optical imaging?

Allowing for detailed analysis of tissue characteristics. (B) Signup and view all the answers

Which optical method utilizes back scattering to obtain images?

Reflection Imaging (D) Signup and view all the answers

Which technique is often used alongside fluorescence for enhanced imaging?

Time gating (C) Signup and view all the answers

What is the primary benefit of confocal microscopy compared to traditional widefield microscopy?

It produces a sharper focus and 3D images. (D) Signup and view all the answers

Which characteristic makes two-photon fluorescence light microscopy suitable for imaging thick materials?

Minimized thermal damage. (B) Signup and view all the answers

What is the functioning principle of Optical Coherence Tomography (OCT)?

It measures echo time-delay and intensity of back-scattered light. (D) Signup and view all the answers

What is a limitation of Optical Coherence Tomography in comparison to ultrasound imaging?

The light cannot penetrate as deeply into the sample. (C) Signup and view all the answers

Why are high optical intensities required in microscopy techniques?

To minimize photo-oxidation of fluorochromes. (B) Signup and view all the answers

What type of laser pulses are typically used in two-photon fluorescence microscopy to reduce thermal damage?

Femtosecond or picosecond laser pulses. (C) Signup and view all the answers

In two-photon fluorescence microscopy, what aspect is specifically focused on to achieve high intensity light in a small region?

Focusing the lens to a single point. (D) Signup and view all the answers

Which of the following is NOT a characteristic of two-photon fluorescence light microscopy?

Requires a pinhole for imaging. (D) Signup and view all the answers

What challenge do traditional bright field illumination techniques face when observing biological samples?

They struggle to detect small amplitude changes in transmitted light. (B) Signup and view all the answers

What phenomenon does light experience when passing through a biological sample due to refractive index changes?

Diffraction, refraction, and phase changes (D) Signup and view all the answers

What is the primary role of the phase plate in phase contrast microscopy?

To change the amplitude and phase of direct light only (D) Signup and view all the answers

In dark-field microscopy, what type of light is primarily collected by the objective lens?

Diffracted/refracted light (A) Signup and view all the answers

Which microscopy technique allows images to be formed without using staining on the sample?

Dark-field microscopy (D) Signup and view all the answers

What typically happens to the amplitude when direct and diffracted light are brought into phase in phase contrast microscopy?

The amplitude increases due to interference (A) Signup and view all the answers

Why may dark-field microscopy require very strong illumination?

To combat low received light intensity (D) Signup and view all the answers

What advantage does phase contrast microscopy offer over traditional microscopy methods?

It improves image contrast using phase information. (B) Signup and view all the answers

What effect occurs when the direct and diffracted light are completely out of phase in phase contrast microscopy?

They cancel each other out, resulting in darkness (B) Signup and view all the answers

Epi-fluorescence microscopy primarily works by exciting the sample with what type of light?

Short wavelength light (A) Signup and view all the answers

What is the main purpose of scanning the reference mirror in optical coherence tomography (OCT)?

To record the intensity of optical signals (B) Signup and view all the answers

How does coherence length affect depth resolution in OCT?

Shorter coherence length leads to better resolution (C) Signup and view all the answers

Which optical component can improve depth resolution in OCT?

A confocal aperture (B) Signup and view all the answers

What is a primary advantage of optical coherence tomography over traditional ultrasound?

Higher resolution imaging of structures (D) Signup and view all the answers

What additional data do spectral and time-resolved imaging techniques provide?

Real-time observation of biological functions (B) Signup and view all the answers

In spectral imaging, what role does frequency content of fluoresced light play?

It allows tracking of multiple fluorescent markers (D) Signup and view all the answers

What mechanism does fluorescence resonance energy transfer (FRET) utilize?

Non-radiative energy transfer between closely placed fluorophores (C) Signup and view all the answers

What is essential for measuring the IA/ID ratio in FRET imaging?

Intensity of the emitted light from both fluorophores (A) Signup and view all the answers

Which aspect of microscope resolution is enhanced by using excited fluorophores?

Both spatial and temporal resolution (B) Signup and view all the answers

What effect does the proximity of donor and acceptor fluorophores have in FRET?

Triggers non-radiative energy transfer (D) Signup and view all the answers

Which microscopy technique is primarily used in conjunction with spectral imaging?

Epi-fluorescence microscopy (B) Signup and view all the answers

What does the size of the optical beam spot on a sample affect in OCT?

Transverse resolution (D) Signup and view all the answers

How can spectral imaging help in drug-organelle interaction studies?

By examining shifts in the emission profiles (C) Signup and view all the answers

What is the significance of the R-6 distance dependence in FRET?

Indicates the maximum distance for energy transfer (D) Signup and view all the answers

Study Notes

Module Structure

Fundamentals of light
Propagation of light in waveguides
Fundamentals of matter
Light interaction with matter
Laser, LED, and photodetector basics
Photobiology basics
Biophotonics applications
Bioimaging
Optical Biosensors
Flow cytometry
Light activated therapy
Tissue engineering

Topics to be Covered

Introduction
Optical and Non-Optical
Overview of methods
Transmission, reflection, fluorescence
Imaging techniques
Phase contrast microscopy
Dark-field microscopy
Fluorescence microscopy
Confocal microscopy
Two-photon fluorescence light microscopy
Optical coherence tomography
Fluorescence resonance energy transfer

Introduction

Bioimaging is a major branch of Biophotonics
X-ray, CT, ultrasound, and MRI are geared towards organ-level imaging
Cellular and sub-cellular level imaging is often required for diagnostics and treatments
Optical techniques allow the study of various specimens (in vivo, ex vivo, in vitro)
Imaging relies on optical contrast in transmission, reflection, or fluorescence between the area of interest and the background

Non-Optical vs. Optical Imaging

Non-Optical Methods: X-ray and CT scans cause ionization, harmful, unsuitable for young patients and cannot distinguish between benign and malignant tumours. MRI cannot provide real-time cellular level changes and resolution in ultrasound is poor.
Optical Methods: Not harmful (above UV), imaging objects as small as 100 nm, multidimensional imaging is possible, imaging of in vivo, in vitro specimens, fluorescence imaging can monitor spectra, quantum efficiency, lifetime and polarization. Optical imaging can be combined with other techniques.

Optical Methods of Imaging

Optical Imaging (categories):
- Transmission (Transillumination)
- Reflection (Back Scattering)
- Fluorescence

Transmission

Tissue is a highly scattering medium
When a sample is illuminated, photons emerge at the end and can be categorized into:
- Coherently scattered (ballistic) photons: shortest route to the detector
- Slightly longer to arrive (snake) photons: undergo severe scattering
- Diffuse photons: longer paths before reaching the detector

Techniques for Transmission Microscopy

Spatial Filtering: Confocal aperture rejects off-axis photons (mostly diffuse photons)
Polarization Gating: Ballistic photons maintain polarization; diffuse photons get partially or completely depolarized
Time Gating: Short pulse of light, optical gate at receiver (allowing ballistic/snake photons). Established techniques exist for synchronizing gate to ballistic photons

Reflection

Reflection imaging collects back-scattered light
Coherent back-scattered light must be discriminated from multiply back-scattered light
Confocal and interferometric techniques are used
Confocal: Spatial filtering using a confocal aperture on central axis
Interferometric (OCT): optical coherence tomography uses interference between a reference signal and back-scattered light.

Fluorescence

Widely used optical bioimaging technique
Detailed probing of structure and dynamics (in vitro and in vivo) for diverse tissue dimensions
High signal-to-noise ratio enables imaging of small samples
Number of fluorophores available for tagging biological samples
Fluorophores bind to specific molecules allowing observation of specific organelles under fluorescence microscopy

Two-Photon Fluorescence Light Microscopy

Simultaneous excitation of molecules by two low-energy (typically IR spectrum) photons
High intensity light is focused into a very small region to enable 3D imaging, eliminating the need for pinholes.
Typically uses femto/picosecond laser pulses to minimise thermal damage and is suitable for thick samples due to less tissue absorption and scattering in the IR

Optical Coherence Tomography (OCT)

Forms reflection images similar to ultrasound imaging; measuring the "echo time-delay" and intensity of back-scattered and back-reflected light
Unlike ultrasound, does not require contact
Higher resolution than ultrasound
Time resolution required to detect light echo in a 10µm sample is in the 30fs range, which is outside the range of modern electronics and requires a different detection method (Michelson Interferometer)

Optical Coherence Tomography (OCT) - 2

Light source splits into two equal beams by a beamsplitter
A reference beam is directed towards a movable reference mirror
Sample beam is directed towards the sample under observation
Reflected beams recombine at the beam splitter and are collected by the sensor
Resultant interference caused is dependent on the difference in travel distances of each beam

Optical Coherence Tomography (OCT) - 3

Interference fringes will be visible at the detector as the reference mirror is moved, changing the reference beam path length
Highly coherent sources (e.g. laser) produce consistent interference fringes irrespective of path changes
Low-coherence sources (e.g. LED) produce detectable constructive interference when path length differences are within the source's coherence length
Coherence length is the distance over which light wave exhibits temporal coherence with shorter length improving resolution
Superluminescent light source is required compared to a laser

Optical Coherence Tomography (OCT) - 4

For a fixed sample, reference mirror is scanned up and down
Intensity is recorded at the detector for various locations
Echo time and magnitude of the backscattered sample beam are measured by scanning the reference mirror
Depth resolution is determined by the coherence length; shorter coherence length leads to better depth resolution, which in turn depends on the source
Transverse resolution is determined by the beam spot size

Optical Coherence Tomography (OCT) - 5

Advantages: High resolution – 4-10 µm (compared with 110 µm of ultrasound), Real-time imaging, Fiber optic designs can be integrated with catheters and endoscopes
OCT of the retina can utilize the technique

Spectral and Time-Resolved Imaging

Current microscopy methods can achieve sub-cellular resolution, however not enough to observe biological function in real time.
Spectral & time-resolved imaging is used for the detection of fluorescence to complement real-time observation of biological processes.

Spectral Imaging

In fluorescence-based imaging, spatial imaging provides cell structural information. Spectral imaging provides additional information by examining the frequency content of the fluoresced light
Spectral imaging allows multiple fluorescent markers to be used & tracked within a sample
Shifts in emission profiles correlate to biological processes, useful in drug and organelle interaction studies. Techniques can involve bandpass filtering, excitation wavelength tuning for marker separation

Fluorescence Resonance Energy Transfer (FRET) Imaging

Spectral imaging technique utilizing two distinct fluorophores (donor and acceptor)
Energy transfer occurs when the emission from the donor overlaps the absorption band of the acceptor if the fluorophores are within ~10 nm of each other
The intensity of emission of each marker is measured at different locations to generate 3D images
This allows for fine resolution imaging less than 10 nm

Fluorescence Resonance Energy Transfer (FRET) Imaging - 2

Imaging is completed by measuring and calculating the ratio of donor (Id) and acceptor (IA) emission intensities at different XY locations in the sample
Dipole-dipole interaction causing non-radiative energy transfer occurs only at very close proximities
Distance dependence of the process falls at a rate of R⁻⁶ (where R is the separation distance)

Summary

Optical imaging techniques are crucial for understanding biological structures and processes non-destructively.
Imaging methods detect transmitted, reflected, backscattered light and fluorescence from a sample
Modern techniques use XY scanning to create comprehensive 2D (spatial) images
Subwavelength resolutions are achievable with near-field optical methods
Spectral and temporal imaging enables the observation of biological functions.

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Description

This quiz covers the fundamentals of biophotonics and bioimaging, focusing on the interaction of light with matter. Key topics include various imaging techniques like fluorescence and confocal microscopy, as well as applications in tissue engineering and diagnostics. Test your knowledge on the principles and methods that enable cellular and sub-cellular imaging.