Skip to main content

Microscopy vs Flow Cytometry



Fluorescence Microscopy and Flow Cytometry, both are biological instruments which are capable of measuring the biophysical and biochemical properties of cells or particles which are under study. Both of them are capable of identifying types of cells present in a given sample. These two technologies are useful in variety of applications and they have widespread usage... But, These two differ in their applications and use. 
Fluorescence Microscopy
Flow Cytometry
Sample mounted on a glass slide.
Sample is allowed to pass through a liquid stream through a sensing area. (Single cell suspension).
Produces magnified images of the cells
Does not produce the images.
Whole Tissue section can be visualized and analyzed
Tissue must be disintegrated into single cell suspension. And each single cell is analyzed.
Detection through human eye and images can be produced on a computer screen
Detection is electronic mode.
Electronic Data.
Xenon or Mercury-Vapor Lamp are used as light sources.
Usually, Lasers are used as light sources or excitation sources.
Sample number of cells are analyzed.
10-100s of cells on a slide
High Throughput analysis.
1000s of cells per second.
Objective Interpretation
Subjective Interpretation
Slow Analysis Time and Less Sensitivity
High throughput and High Sensitivity
Qualitative Results
Quantitative Results


Note: Both systems use Dichroic Mirrors and Emission Filters.

Fluorescence Microscopy: Microscope was invented before flow cytometry, fluorescence microscopy is improved version of a light microscope, uses the lasers as excitation sources to excite the fluorescence present in the specimen under study. Fluorescence emitted from the cells or particles present in specimen produces a magnified image. Fluorescence Microscope is useful to study the specific cell characteristics. With the help of this, cells can be quantified and can be analyzed qualitatively. However, number of cells or types of cells are limited. 



Flow Cytometry: with the help of this technology, cells or particles present in a suspension can be quantified and analyzed and perform measurement for the characteristics such as relative cell size, granularity and quantification of DNA & RNA, fluorescence and intracellular proteins. Results obtained from the flow cytometer could be absolute or relative data. The technology has been widely used for the identification and characterization of cells, cell types, cell counting, and identifying various extra and intra cellular protein markers, identification and characterization of micro organisms. 

Fluorescene Microscopy vs Flow Cytometry: 
       Fluorescence Microscope and Flow Cytometer, both are capable of measuring the cellular components quantitatively. But, they differ in how many cells they can handle, locating the cellular components, volume of the cells, number of parameters and also sample preparation. 

Fluorescence Microscope can show how the cellular components are distributed inside the cell (clustered or uniformly). It can also show the area of the cell and its structure. It can quantify the cellular components for 10-100s of cells and one parameter at a time is used. This technology produces magnified images of the cells. Sample preparation involves the mounting the specific number of cells on a slide and observe under the microscope to produce a magnified image. One fluorescence parameter can be used simultaneously. Can not sort the cells. 

But, where as 

Flow cytometer can not show the distribution of cellular components within the cells, But it can measure the fluorescence tagged with those cellular components and measure them quantitatively. It can produce relative data of the cell size,  cell granularity and other properties such as fluorescence data. It can handle up to 1000s of cells per second. (There is no magnified image). Sample preparation involves the preparation of single cell suspension and maintaining these cells dispersed in the suspension. Up to 15-24 fluorescence parameters can be used simultaneously. Also, target cells can be sorted using the technique called fluorescence-activated cell sorting and sorted cells can be further used for downstream applications. 


"Flow Cytometer and Fluorescence Microscope achieve same goals. They have their own roles, benefits, advantages and applications. Due to these reasons, it makes them suitable for the different specific roles" 


Labels:

Comments

Post a Comment

Popular posts from this blog

Statistics in Flow Cytometry Data and "MFI" values

                                 The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity..                Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values.  (Keeping in mind that, consideration of statistics in flow cyomtery also depends on the type of Application you are interest
Post a Comment
....CONTINUE READING

Gene Cloning Technique

Gene Cloning Technique in Molecular Biology Field Involves the following steps: 1.a) Isolation of Genetic Material and Gene Sequence of Interest.  First, cells are lysed using detergent or lysozyme enzymes which disrupts the plasma membrane and release the genetic material along with the macro molecules such proteins and RNA molecules. Cell contents are then treated with protease to disrupt the proteins and RNase to destroy the RNA. Cell debris are then pelleted using centrifuge and supernatant containing DNA is transferred to a fresh and clean tube. A proper amount of Ethanol is added to this supernatant and precipitated using centrifuge. Supernatant is discarded and the pellet which has DNA is suspended using a proper suitable buffer.  Primers are designed for the specific gene sequence of interest which will be used for the cloning procedure.  Gene sequence is then amplified using PCR (Polymerase Chain Reaction) which will yield in many copies of the gene sequence.
Post a Comment
....CONTINUE READING

Nanodrop

Thermofisher, Nanodrop 1000    Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm).  Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument.  It works on the principle of Spectrophotometer or Sample Retention System....  Principle of Nanodrop: "The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of column and forms approxi
Post a Comment
....CONTINUE READING