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Confocal Microscopy: Principles & Experiments

OBJECTIVE: In traditional wide field fluorescence microscopy, we cannot get image of high quality for thick specimen. In confocal microscopy, a pinhole is used to get image of the focus by rejecting the out of focus lights, and hence we can have images of better resolution and contrast of thick samples.

THEORY: The whole light coming out of a fluorescent sample is collected in the case of traditional wide filed microscopy whereas, in the confocal microscope, first the image light is focused onto a point (optically conjugate plane of the image), and there a pinhole of proper size is placed to get rid of the light coming from the other portion of the sample and thus getting the image of the focal plane only. Here, first a Continuous Wave (CW) laser light is focused on the fluorescent sample using microscope objective to excite the sample. Then the fluorescence coming from the sample is collected by the same microscope objective. The fluorescence signal is separated through a dichroic mirror and then focused and defocused before it goes to the detector and at that focal point, a pinhole called confocal aperture is placed to remove the fluorescence generating from outside of the focal spot region.


Schematic of Confocal Microscopy


  1. Excitation Laser
  2. Microscope
  3. Microscope Objective
  4. Fluorescent Sample
  5. Dichroic Mirror
  6. Lenses
  7. Pinhole
  8. Detector



Note: For user operation and usage no specific software needed.


1.      Turn on the excitation CW Laser.

2.      Place a drop of immersion oil on the oil immersion microscope objective of a specific magnification.

3.      Place the microscopic slide of stained live cell on the sample plane.

4.      Tighten it with the clamps.

5.      Turn on the UV lamp.

6.      Move the objective such that the oil placed on it touches the bottom side of the slide.

7.      Let the UV light pass through the objective and get focused on the sample (i.e., microscopic slide) and see the fluorescence coming from the stained live cell through the microscope eye piece.

8.      Focus the sample seeing the fluorescence of the cell(s).

9.      Block the UV lamp.

10.  Turn on the FLUOVIEW software for image collection.

11.  Fix the power of the excitation Laser beam.

12.  Let the excitation Laser beam pass through the microscope objective.

13.  Set the value of confocal aperture according to the magnification of the microscope objective (e.g., 40X, 60X).

14.  Place proper fluorescence filter (i.e., band pass filter) for selecting the fluorescence coming from the specific region of the cells (e.g., nucleus, microtubules etc.).

15.  Turn on the Photo Multiplier Tube (PMT) detector to collect the fluorescence.

16.  Increase the PMT voltage to have a good signal.

17.  Select the area of the cell to be scanned for getting the image.

18.  Click on the ‘scan once’ button getting the image of the live cell sample.

19.  Save the image by clicking the buttons in the following order: File I/O => Save Image as and then select the file type (.TIFF or .BMP) and where to save the image and then click the ‘save’ button to save the image(s).


Confocal image of BPAE tissue