10 Difference between Fluorescence Microscope and Confocal Microscope

Fluorescence Microscope and Confocal Microscope Comparison

First let us begin with the similarities. Both Fluorescence and Confocal Microscope uses use fluorescent molecules to image specimens.

In fluorescence microscopy, a specimen is illuminated with a light source that excites fluorescent molecules in the specimen. The emitted light from these molecules is then detected and used to create an image.

In confocal microscopy, point illumination source or a focused laser beam is used to illuminate the specimen and the emitted light is passed through a pinhole; to eliminate out-of-focus light. This allows for higher resolution images than traditional fluorescence microscopy.

Both can be used to image live cells and thick tissues. Both these microscopes are widely used in live cell imaging, biomedical research and drug discovery.

Fluorescence Microscope	Confocal Microscope
Principle: Illuminates the entire specimen with a broad light source and the emitted light from these molecules is then detected and used to create an image	A point illumination source is used to scan the specimen, and the emitted light is passed through a pinhole; to eliminate out-of-focus light
Lower resolution	Higher resolution, especially in the z-axis as out of focus light is eliminated
Deeper depth of field, can image thick specimens	Very shallow depth of field that is only a very thin slice of the specimen is in focus at any given time
Does not use a confocal pinhole.	Uses a confocal pinhole to reject out-of-focus light.
Can image multiple structures with a single fluorescent label (single labeling).	Requires multiple fluorescent labels (multiple labeling) to image multiple structures like chromosome, spindle fibers etc
Acquires images all at once, which is faster.	Acquires images point-by-point, which can be time-consuming.
3D imaging possible but not fine as confocal microscope	3D imaging possible with high resolution
Imaging of large specimens, such as whole cells or tissues	Application: Imaging of thick tissues, live cells, and single molecules
Can be used for quantitative analysis, but effective as confocal microscopy.	Can be used for quantitative analysis like no. of cells, size of cell structures etc.
Less expensive	More expensive

Image credit: https://www.olympus-lifescience.com/

Watch our simplified video on Types of Microscope

Advantages of confocal microscopes:

• Higher resolution
• Shallow depth of field
• Ability to image thick tissues and live cells
• Good for imaging single molecules

Advantages of fluorescence microscopes:

• Less expensive
• Can image thicker specimens
• Good for imaging large specimens

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