Confocal Microscopy

Authors: Maura Francis, Cory Boone, Jaclyn Wycoff

Confocal microscopy is a type of fluorescence microscopy which uses a laser to excite fluorescence from fluorophores used to label different subsets of a specimen. Fluorescence microscopes are used for imaging cells and tissues that have been fluorescently tagged.¹ What separates confocal microscopy from conventional epi-fluorescence microscopy is its increased resolution when imaging thick specimens, elimination of out-of-focus glare due to spatial filtering, and reduction of light-induced damage to the sample, known as phototoxicity.

Instead of using an incoherent tungsten or mercury lamp source like conventional microscopes, confocal microscopes use a laser to illuminate the sample.¹ The system then collects images at planes at various depths in the sample, known as optical sections, by scanning the laser through different focus positions.¹ Optical sections enable live specimens to be imaged because the sample doesn’t need to be physically sectioned off. The illumination of smaller sections also helps specimens to remain viable longer by significantly reducing the effects of phototoxicity. The amount of light that conventional techniques use for illumination makes it difficult to ensure the vitality of the specimen imaged, which is crucial when capturing biological events.¹

Image Appearance

When compared to images captured by a conventional fluorescence microscope, images from a confocal microscope system exhibit a marginal increase in axial and lateral resolution, but spatial filtering from the system’s pinhole assembly blocks out-of-focus fluorescence for a much more detailed image (Figure 1).² Contrast of the image is improved over traditional methods due to a reduction in background noise and improved signal-to-noise ratio. When using conventional techniques, the fluorescence emitted from adjacent parts of the sample interferes with the image plane and obscures the focus.¹ This is problematic because there is no way of differentiating the fluorescence that does not contribute to the focal plane being imaged, which is especially apparent in specimens that are thicker than two micrometers.¹ By utilizing spatial filtering techniques, confocal microscopes are able to eliminate this problem by blocking any out-of-focus light that would otherwise obscure the image.² Confocal and multiphoton microscopy have similar resolution in the x- and y-axes, but multiphoton microscopy has superior resolution in the z-axis, or depth. Compared to multiphoton microscopy, confocal microscopy only uses a single photon to excite fluorescent dyes, which can result in more light scatter in the image and the inability to image as deep into a sample. Confocal techniques commonly use light from the visible spectrum, which also tends to scatter and absorb more in biological tissue than longer wavelengths, which in turn can limit the possible penetration depth into a sample. However, the confocal technique still has proven to have enhanced resolution over traditional widefield techniques, allowing it to be considered a bridge between classic widefield methods and more advanced super-resolution techniques. Confocal microscopy is very well-suited for imaging of 2D cell cultures while multiphoton and light sheet microscopy provide advantages for imaging 3D samples.

Figure 1: An image of protoplasts captured using confocal microscopy (left) that is more finely focused than an image of microspheres of a similar size captured using conventional epi-fluorescence microscopy.

Technical Details

Figure 2 depicts the typical layout of a confocal microscope in which there are four key components:

Laser Excitation Source: emits a laser beam into the system to focus on the sample.
Fluorescence Dichroic Filters: reflects the excitation light onto the specimen and passes the secondary fluorescence, given off by the specimen, through to the detection system.
Pinhole: an essential component of confocal setups, it acts as a spatial filter. It prevents any light that is not confocal to the objective focal plane from interfering with the image.²
Stepper Motors: allows the laser beam to travel incrementally across the sample, obtaining x and y scans collected along the z-axis. This automates three-dimensional data being collected, making it easy to create a 3D image.²
Objective: a high numerical aperture (NA) objective is typically used to achieve high resolutions. Water or oil immersion designs are often used to adjust for refractive index mismatch between the aqueous medium many live samples are kept in. Long working distances are required for thick specimens and 3D imaging/optical sectioning.

Figure 2: Schematic of a typical confocal microscopy system

Comparison to Other Microscopy Techniques³

Technique	Resolution	Sample Size	Relative Cost	Photobleacing
Confocal Microscopy	<µm	µm	$$	Yes
Multiphoton Microscopy	<µm	mm	$$$	Less
Light Sheet Fluorescence Microscopy	µm	>cm	$	Least

Confocal Microscopy at Edmund Optics®

Edmund Optics® supplies a wide range of optics for confocal fluorescence microscopy applications including filters, objectives, laser systems, and pinholes.

Fluorescence Filter Sets

Excitation, Emission, and Dichroic Filters for Fluorescence Imaging
>93% Transmission and OD 6 Blocking for Maximum Brightness and Contrast
Hard Sputtered Coatings on Single Substrate

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Pre-Mounted Fluorescence Filter Cube Sets

Ensure Proper Alignment of Filter Sets
Easily Adapt to Common Microscopes
Available in a Variety of Wavelengths

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Fluorescence Bandpass Filters

Common Wavelengths for Popular Fluorophores
Excitation Filters and Emission Filters Available
>93% Transmission
>OD 6 Blocking, <3% from Edge of Bandwidth

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High Performance Fluorescence Dichroic Filters

Improved Flatness, Transmitted Wavefront, and Surface Quality
Ideal for Fluorescence Microscopy or High Magnification Imaging Applications
Most Popular Cut-On Wavelengths of Fluorescence and Longpass Dichroic Filters

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Olympus Water Immersion Objectives

Water Immersion Objectives with Magnification Ranges from 10 to 40X
Displays Flat Images from High Transmission Factors up to the Near-Infrared Region of the Spectrum
Ideal for Fluorescence Imaging of Tissue and Specimens, such as Brain Tissue

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Olympus X-Line Extended Apochromat Objectives

High Numerical Aperture (NA) up to 1.45
Chromatic Aberration Correction from 400 - 1000nm
Uniform Image Flatness over Large FOVs

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Mitutoyo Infinity Corrected Long Working Distance Objectives

Long Working Distances
Bright Field Inspection
High Quality Plan Apochromat Design
Flat Image Surface over Entire Field of View

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Coherent^® High Performance OBIS™ LX/LS Laser Systems

Same Compact Design for All Wavelength Options
Integrated Control Electronics with Analog and Digital Modulation
Circular Beam with Superior Beam Quality

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Coherent^® High Performance OBIS™ LX/LS Fiber-Pigtailed Laser Systems

High Performance OBIS™ LX/LS Lasers with Added Fiber Optic Capability
Permanent Fiber Attachment Extends Lifetime with Guaranteed Power
Single-Mode Polarization-Maintaining Fiber with an FC/APC Connector Provide High-Quality and Low-Noise Laser Beam Output

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Precision Pinholes

Available in Aperture Mounts for a Secure Mechanical Support
Pinhole Sizes Ranging from 1 to 1,000 Microns
High Power Apertures Available

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Acktar Blackened Pinholes

Low Reflectance from the EUV to SWIR
99% Absorbance Blackening Coating
Aperture Sizes from 20 to 1000μm
Available Unmounted and Mounted

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References

Paddock, Stephen W., et al. “Introductory Confocal Concepts.” Nikon, https://www.microscopyu.com/techniques/confocal/introductory-confocal-concepts.
Fellers, Thomas J., and Michael W. Davidson. “Introduction to Confocal Microscopy.” Olympus Scientific Solutions Americas Corp., https://www.olympus-lifescience.com/ru/microscope-resource/primer/techniques/confocal/confocalintro/.
Santi, P. A. (2011). Light Sheet Fluorescence Microscopy. J Histochem Cytochem, 59(2), 129-138. doi: 10.1369/0022155410394857.