Overview

Confocal and multiphoton are modern fluorescence techniques in microscopy for generating optical sections from live or fixed biological specimens. In general, both techniques employ a point scanning and point detecting design. The confocal microscope achieves point detection by using a confocal pinhole to block off out-of-focus emission from the specimen. The multiphoton microscope, on the other hand, generates intrinsic point emission directly from the in-focus spot, thereby eliminating the need for a confocal pinhole. Subsequent scanning of the entire field of view results in an optical section. Computer reconstruction of serial optical sections, collected at consecutive axial (z) positions, can reveal the spatial localization of cells and tissues (sometime subcellular molecules) in 3D. The ability to see biochemical processes in live cells, in real time, sheds light on the vastly complex molecular world of cells and may allow scientists to identify new targets for drugs that will treat exposure to dangerous toxins and bacteria as well as fight a wide variety of diseases.  The table below summarizes and compares some of these technologies that are available here at PCMM.

 

Digital Deconvolution

Spinning Disk Confocal

Standard (point scan) Confocal

Multiphoton

General principles

Uses computer algorithm to estimate the characteristics of light from in/out of focus planes to remove all out of focus signals leaving an in focus image (resulting in "confocal like" optical sections).

Multiple point illumination (pinholes from a disk) and same multiple point detection to block out of focus light. Image obtained instantaneously when disk spins at high speed.

Single point illumination and same point detection to block out of focus light (one point at a time). Image built up after scanning the area.

Single point illumination with intrinsic optical sectioning (emission arising from focal point only). Image built up after scanning the area.

Speed

Slow (10 min to hrs)

Fast (real time; video rate - 1/30 sec or faster per image)

Varies (non real time; ¼ sec to 2 sec per image)

Varies (non real time; ¼ sec to 2 sec)

Imaging depth in tissue (approx.)

n/a

20 µm

100 µm

250 µm +

Light source

n/a

White light; arc lamp; laser

Laser

Laser

Phototoxicity

n/a

Low

High

Very low

Uniqueness

It is a software that works on conventional microscopes. In certain cases, it can also be applied to confocal and multiphoton images as a post-processing image enhancement tool.

Able to capture dynamic events lasting only fractions of a second (common in live cell applications).

Optical zoom; precision dissection and photobleching; cross sections

Optical zoom; precision dissection and photobleching; cross sections; deep tissue imaging; low phototoxicity

General comment

Less useful as standalone but compliment well with confocal and multiphoton

Flavored for live cell imaging

Overall multifunctional workhorse

For deep tissue imaging; indispensable for intravital work

Availability in PCMM Core

Yes

Yes

Yes

Yes