Light microscopes are essential tools for magnifying tiny structures and details invisible to our naked eyes. However, not all microscopes are alike – they utilize different illumination techniques to image samples. This article compares two major forms: transmitted light microscopy and reflected light microscopy.
Transmitted light microscopes allow light as a form of electromagnetic radiation, to pass through thin samples, creating a contrast to see internal composition and structure. Reflected light microscopes, on the other hand, bounce light off the sample surface to analyze topography and exterior features.
The remainder of the article explains the key differences between these modes of light microscopy in depth, including how each one works, their respective applications and limitations, and when one is preferred over the other. Read on to understand how to select the optimal microscopic illumination approach for your research needs.
How Transmitted Light Microscopes Work
Transmitted light microscopes, also known as brightfield microscopes, use light that is transmitted through the sample being observed.
The light source, typically an LED or halogen bulb, sits below the sample stage and projects light upwards through a condenser lens, which focuses the light on the sample. The light passes through the sample and is then captured by the objective lens. The objective lens magnifies the image of the sample. This magnified image then passes through the ocular lens where it can be viewed by the eye or projected onto a screen or camera.
As the light passes through the sample, some of it gets absorbed or scattered. This creates contrast in the sample that allows internal structures to be visualized. Thicker areas or areas with higher density absorb more light and appear darker, while thinner or less dense areas allow more light to pass through and appear lighter. This makes transmitted light microscopes ideal for viewing the internal composition of transparent specimens.
Applications of Transmitted Light Microscopy
Transmitted light microscopes excel at imaging thin and translucent samples. Some common applications include:
- Biological samples – Cells, microorganisms, thin tissue slices, and microbiological cultures can be imaged with high contrast and resolution. Staining samples with dyes or fluorescence tags provides additional contrast.
- Mineralogy and petrology – The internal structures of minerals and rocks can be analyzed. Thin sections are often prepared.
- Semiconductors – Integrated circuit components and defects can be inspected.
- Forensics – Hair, fibers, and gunshot residues are examples of trace evidence that can be analyzed.
Limitations of Transmitted Light Microscopy
The main limitations of transmitted light microscopy include:
- Samples must be very thin and translucent. Thick or opaque samples will not allow light to pass through.
- Resolution is limited by diffraction to 0.2 – 0.3 microns.
- Out-of-focus light outside the focal plane can reduce image contrast.
- Additional sample preparation like thin sectioning may be required.
- Staining or fluorescence tagging is often needed to introduce contrast.
- Living organisms may be adversely affected.
How Reflected Light Microscopes Work
In contrast to transmitted light microscopes, reflected light microscopes use illumination that reflects off the surface of the sample rather than passing through it.
The light source provides incident light that reflects off the sample surface. This reflected light enters the objective lens where it is gathered and focused into an image. At the beam splitter, some percentage of light is deflected towards the ocular lens and camera/eyepiece.
Reflected light microscopes allow samples to be viewed directly without specialized preparation since light does not need to transmit through the sample. Topographic and compositional details of the sample surface can be visualized.
Applications of Reflected Light Microscopy
Some examples of where reflected light microscopy excels include:
- Semiconductors and electronics – Highly detailed imaging of circuits and microchip topography. Defect inspection.
- Metallurgy and materials science – Analysis of alloys, composites, coatings, and fractures.
- Geology – Characterization of rocks, minerals, and soils.
- Forensics – Imaging of textile fibers, documents, and ballistics.
- Quality control and manufacturing – Surface inspection for defects in industrial parts and products.
Limitations of Reflected Light Microscopy
Limitations of reflected light microscopy include:
- Provides no information about subsurface composition and structure. Only the surface is imaged.
- Significant image artifacts can occur at high magnification due to angled illumination.
- Maximum resolution is limited by diffraction and optical aberrations.
- Generally lower resolution than transmitted light microscopy.
- Only useful for imaging opaque, reflective, and 3D samples.
Key Differences Summarized
|Transmitted Light Microscope
|Reflected Light Microscope
|Light reflects off the sample surface
|Additional sample preparation is often needed
|Ideal for thin, translucent samples
|Ideal for thick, opaque samples
|Shows internal structure and composition
|Shows surface topography and features
|Higher maximum resolution
|Lower maximum resolution
|Additional sample preparation often needed
|Minimal sample preparation needed
When would reflected light microscopy be preferred over transmitted microscopy?
Reflected microscopy is preferred for imaging opaque, thick, or 3D samples where light cannot transmit through. This includes inspection of circuit boards, fractures in materials, forensic samples, and surface quality control. Reflected microscopy requires little to no sample preparation.
What limits the maximum resolution in transmitted vs reflected light microscopy?
In transmitted light microscopy, resolution is limited by diffraction to around 200-300 nm. Resolution is dependent on numerical aperture and wavelength. In reflected light microscopy, additional factors like illumination angle and optical aberrations limit the maximum resolution to around 500-1000 nm generally.