10 Questions
What is the purpose of the fluidic system in a flow cytometer?
To present samples to the interrogation point and take away waste
What does the magnitude of the voltage pulse recorded for each cell represent?
Cell size
What is the primary purpose of the detectors in a flow cytometer?
To receive the light signals and convert them into digital data
What is the difference between forward scatter and side scatter?
Forward scatter measures cell size, while side scatter measures cell complexity
What is the purpose of using fluorophore-labeled antibodies in flow cytometry?
To label cell markers and analyze cellular characteristics
What is the purpose of the bandpass filters in a flow cytometer?
To collect discrete fluorescence data for specific fluorophores
What is the primary function of the forward scatter threshold in a flow cytometer?
To prevent the domination of data by small particles and debris
What is the outcome of the fluorescence emission in a flow cytometer?
A voltage pulse proportional to the amount of fluorescence emitted
What is the purpose of the detector in a flow cytometer?
To translate the fluorescence into a voltage pulse
What would happen if the forward scatter threshold was not set in a flow cytometer?
The data would be dominated by small particles and debris
Study Notes
Flow Cytometry
- Flow cytometry is a powerful technique for analyzing multiple parameters of individual cells within heterogeneous populations.
Instrumentation
- A flow cytometer consists of:
- Fluidic system: presents samples to the interrogation point and takes away waste
- Lasers: the light source for scatter and fluorescence
- Optics: gather and direct the light
- Detectors: receive the light
- Electronics and peripheral computer system: convert signals from detectors into digital data and perform necessary analyses
Forward Scatter Histogram
- Forward scatter measures cell size
- The scattered light received by the detector is translated into a voltage pulse
- The magnitude of the voltage pulse recorded for each cell is proportional to the cell size
- A histogram of forward-scatter data is a graphical representation of the size distribution within the population
Side Scatter Histogram
- Side scatter measures granularity and structural complexity inside the cell
- The signals collected by the side-scatter detector can be plotted on one-dimensional histograms
- Using two-dimensional dot or scatter plots, one can correlate size and complexity of cell populations
Example: 2D Scatter Plot
- Lymphocytes: small cells possessing low internal complexity
- Monocytes: medium-sized cells with slightly more internal complexity
- Neutrophils and other granulocytes: large cells that have a lot of internal complexity
Fluorescence
- Cellular characteristics can be analyzed using flow cytometry involves the use of fluorescent molecules such as fluorophore-labeled antibodies
- The fluorescent light is directed to the appropriate detector where it is translated into a voltage pulse proportional to the amount of fluorescence emitted
- Different fluorophores (e.g. Alexa Fluor® 488 and R-PE) are detected using specific bandpass filters (e.g. 530 nm and 585 nm)
Forward Scatter Threshold
- A threshold is set to exceed a certain forward scatter pulse size to prevent the data pool from being dominated by minute particles like platelets and debris
This quiz covers the basics of flow cytometry, a powerful technique for analyzing multiple parameters of individual cells within heterogeneous populations. Learn about the instrumentation and data analysis in clinical laboratory settings.
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