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Fluorescence Microscopes

How Fluorescence Microscopy Works & How We Save You Time & Money

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Fluorescence microscopy diagram

Microscopes are found in almost all fields of science, and are used to make small objects more easily visible to the naked eye. Depending on the type, they are used in various areas, from general biology to quantum physics.

 Microscope, lens, and sample dish

Microscopes are organized and grouped based on what method of magnification they use. Optical microscopes, for example, use light and a series of mirrors to produce a magnified image of a sample. The two main categories of optical microscopes are simple and compound.

The difference between simple and compound microscopes is that simple microscopes only use one mirror, while compound microscopes use multiple. The most common optical microscopes use visible light as the light source. However, microscopy utilizes other light spectrums as well.

Fluorescence refers to the emission of light due to the absorption of light. When a material with GFP or other fluorescent dye is exposed to electromagnetic radiation, it absorbs that radiation and re-emits some of it. The time difference, less than a millisecond, between when the organic or inorganic object absorbs the radiation and emits it is short due to the photons’ inherent absorption and emission time.

The object will stop emitting fluorescent light once it stops being hit by radiation. When the materials emit light after the radiation source has gone, it is called phosphorescence. The fluorescent objects’ re-emitted light from the fluorochromes has lower energy and thus a lower wavelength signature, making it exploitable in different analytical techniques.

An example of fluorescence can be seen when one uses a blacklight, and previously unseen colors suddenly are illuminated. Other common examples of autofluorescence include minerals and various bioluminescent animals and plants like algae, fish, and insects.

A fluorescence microscope is a type of light microscope that exploits fluorescence and phosphorescence to identify and observe specific microscopic objects. The area of the material to be observed is dyed with fluorophore tags that will illuminate only the intended parts of the sample.

The specimen is struck with electromagnetic radiation of a specific excitation wavelength that is subsequently absorbed by the fluorophores, which then fluoresce radiation with longer wavelengths than the incident light. This difference in wavelengths can then be used to filter out the light from the light source and only analyze light from the fluorescent sample by using a spectral emission filter.

Detailed imaging of live cells, antibodies, and more can be easily achieved using the fluorescence microscope with proper microscopy techniques. The main components that make up these imaging systems include:

  • Light source: Common types are xenon arc lamps and mercury vapor lamps
  • Excitation filter: Filters out undesirable specific wavelengths from a light source.
  • Dichroic mirror: Filter out specific spectra of light or colors. Also called interference filters or accurate color filters.
  • Emission filter: Filters out non-fluorescent light spectra to analyze the desired fluorescence.

Different filter cubes and beam splitters are chosen depending on the specific fluorescent protein used to label the specimen. Single fluorophores are imaged at one time, meaning that a narrow wavelength or one “color” that is emitted from the sample is observed at one time.

High-resolution multi-colored images can be composed by sewing multiple one-color images together. Understanding how to properly perform your fluorescence imaging is critical to fluorescence microscopy since running a sample too many times can cause photobleaching, causing the fluorescent molecules to stop responding.

Fluorophore Imaging Types, Methods, & Cost

A hand holding a microscope lens with an eye drawn on

Epifluorescence

The most widely used type of fluorescent microscope is epifluorescence or epi-illumination microscopes.

Most microscopes have light travel from the light source, through the sample, and into the objective lens where the image magnification occurs, deriving the refractive index. This means the light source shines from beneath the sample. In epifluorescence microscopy, the specimen is illuminated from above. This means that the emitted fluorescence and the illuminating light both travel through the same objective lens.

Due to the excitation light traveling through the objective lens to get to the sample and not the other way around, less diffraction occurs overall, meaning less light needs to be filtered out to isolate the fluorescent light.

Total Internal Reflection (TIRF)

A problem that can occur in fluorescence microscopes is that in thicker objects, the light fluorescing from the background matter will drown out the light fluorescing from the object’s surface.

TIRF microscopes are used to focus on just these surface-bound fluorophores’ emitted light. This is done by using evanescent waves for excitation of the fluorophores rather than direct light from a lamp. Evanescent fields or waves are spatially concentrated in the area where they occur.

This means that when these waves occur, they only excite the fluorophores on the sample’s surface. TIRF’s sub-micron surface selectivity makes it the primary method for single-molecule detection due to its specificity.

Super-Resolved Fluorescence Microscopy

For a long time, most of the scientific community accepted the fact that optical microscopy would never be used to observe nano-dimensional objects. The conventional wisdom was that the limitations on the resolution of wavelengths that can be obtained would limit the usefulness of optical microscopy.

The 2014 Nobel Laureates in Chemistry, however, has completely changed this. Eric Betzig, William Moerner, and Stefan Hell were all awarded the Nobel Prize in Chemistry “for the development of super-resolved fluorescence microscopy.” Their discovery of super-resolution microscopy has brought optical microscopy into the nano dimension, making visualization at the 250-nanometer range simple.

Stimulated Emission Depletion Microscopy

In 2000, Stefan Hell developed stimulated emission depletion (STED) microscopy, which utilizes two lasers to achieve nano-level microscopy. One laser is used to fluoresce specific molecules, while the second is used to cancel out undesired wavelengths.

Eric Betzig and William Moerner developed single-molecule microscopy, which allows them to turn molecules’ fluorescent qualities on and off again. Using multiple images with different molecules, either on or off, they can also achieve nano-level microscopy. These are revolutionary techniques that allow scientists to take a better look at the world around us.

Nanoscopy techniques are now being used to look at synaptic behavior between nerve cells in the brain to better understand diseases like Alzheimer’s, Parkinson’s, and Huntington’s disease.

We Offer TIRF Fluorescence Microscope Leases to Fit Every Need

An animated cylindrical piece of a microscope

Founder-Friendly Leases

Our lease agreements are founder-friendly and flexible, helping you preserve working capital, strengthen the cash flow of your business, and keep business credit lines open for expansions, staffing, and other crucial operational expenses and business development opportunities.

2-5 Year Lease Lengths

Leases range from 2 to 5 years. Length will depend on several factors, including how long you want to use the equipment, equipment type, and your company’s financial position. These are standard factors leasing companies consider and help us tailor a lease agreement to fit your needs.

Your Choice of Manufacturer

We don’t carry an inventory. This means you’re not limited to a specific set of manufacturers. Instead, you can pick the equipment that aligns with your business goals and preferences. We’ll work with the manufacturer of your choice to get the equipment in your facility as quickly as possible.

Maintenance & Repair Coverage

Bundle preventive maintenance and repair coverage with your lease agreement. You can spread those payments over time. Easily maintain your equipment, minimize the chances something will break down, repair instrumentation quickly, and simplify your payment processes.

End-of-Lease Options

At the end of your lease, you have multiple options. You can either renew the lease at a significantly lower price, purchase the machine outright based on the fair market value of the original pricing, or call it a day and we’ll come the pick up the equipment for you free of charge.

No Loan-Like Terms

Our leases do not include loan-like terms, which can be restrictive or harmful in certain situations. We do not require debt covenants, IP pledges, collateral,  or equity participation. Our goal is to maximize your flexibility. When you lease with us, you’re collaborating with a true business partner.

In-House Underwriting Process

Our underwriting is done in-house. You can expect quicker turnaround, allowing you respond to your equipment needs as they arise. We require less documentation than traditional lenders and financiers and can get the equipment you need in operation more quickly.

Popular Fluorescence Microscope Manufacturers

Am Scope
Auxilab
Bruker
Keyence
Leica Microsystems
Luxendo
Molecular Devices
Motic
Olympus
Thermo Fisher Scientific
Zeiss
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