SFA vs high-end systems for stereo fluorescence microscopy

Posted On: Friday, May 8, 2015

We are often asked how our Stereo Microscope Fluorescence Adapter compares with conventional systems for stereo fluorescence microscopy. By ‘conventional’ people typically mean a fluorescence stereo microscope from one of the major microscope suppliers – Nikon/Olympus/Zeiss/Leica (abbreviated NOZL from here on), and they are asking ‘How does something look under yours vs theirs?’.

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There is no simple quantitative answer that covers all cases, but we can make a quick generalization:

• If your specimen exhibits reasonably bright fluorescence and is easy to see on a NOZL system then the NIGHTSEA SFA system will almost certainly work.
• If you are having a hard time seeing the fluorescence (very small, very weak) on a NOZL system then our system is likely not the answer (although of course we would love for you to try ☺).

Feedback from our users indicates that the SFA is ‘just as good’ or ’90 – 95% as good’ for numerous applications. There are also cases in which fluorescence seen under a NOZL system isn’t visible with the SFA. As with anything in microscopy, the results are very application dependent and the typical fluorescence parameters apply: performance depends on how much fluorescing material is present, how brightly it is fluorescing, and how much background there is. Size alone is not always an issue. We have seen individual GFP-expressing thrombocytes, about 5 micron diameter, circulating in the tail of a zebrafish using the SFA system.

A better question?

Probably a better question is ‘Will the SFA do the job that I need to do?’ or ‘Can I see what I need to see?’

NIGHTSEA’s SFA is an excellent choice if you want to:

• Screen/sort transgenic organisms that have a reasonable fluorescence expression;
• Pre-screen samples before scheduling time at a core imaging facility for fluorescence or confocal microscopy;
• Conduct microinjection/microdissection/microsurgery;
• Update stereo microscopes that you already own to make fluorescence more routinely available;
• Outfit multiple stereo microscopes for fluorescence for an undergraduate teaching laboratory;
• Relieve demand on your NOZL fluorescence microscopes for more routine, repetitive investigations;
• Take fluorescence microscopy on the road for demonstration or educational outreach;
• Take fluorescence microscopy into the field to explore fluorescence in nature.

NOZL systems are a better choice if you need to:

• Image weak fluorescence;
• Very rapidly switch between excitation wavelengths;
• Take very high resolution photographs of fluorescence from small features or objects;

How the NOZL and SFA systems differ in design

Differences in performance are coupled to differences in design, so let’s examine those differences in a bit more detail. The two main elements of any fluorescence system are excitation and emission. A third element is generally a filter shield to screen the user from the intense excitation light directed at the sample.

NOZL systems use coaxial epifluorescence

• Excitation
  • typically a high intensity mercury arc source or a multi-wavelength LED source
  • interference filter to select desired excitation wavelength range
  • excitation light integrated with system optics to pass through the objective lens, illuminating the sample from above
• Emission
  • interference filter to select desired emission wavelength range
• Shield
  • generally orange to red to block all of the commonly used excitation wavelengths and protect against any stray ultraviolet

NIGHTSEA Stereo Microscope Fluorescence Adapter

• Excitation
  • interchangeable LED (light emitting diode) light heads, each emitting a dedicated excitation wavelength range
  • light is delivered directly to the sample, rather than through the microscope optics
• Emission
  • interchangeable barrier filters matched to each light head
  • filters magnetically mount beneath the objective lens for quick and easy exchange
• Shield
  • interchangeable shields matched to the barrier filters

In the NOZL systems the fluorescence is tightly integrated with the microscope, while the NIGHTSEA SFA is designed as a near-universal adapter to work with virtually any existing stereo microscope, including both Greenough and CMO designs.

Features and benefits of each approach

Now let’s look at some of the characteristics that users have expressed interest in and compare the systems.

Efficiency in delivering excitation light to the subject

• The NOZL coaxial epifluorescence design is more efficient. Since the excitation light comes through the lens it adjusts along with any changes in the settings of the microscope optics.

Rapid switching between excitation/emission wavelength combinations

• The filter holders in a NOZL system enable near-instantaneous switching between wavelengths.
• Switching between wavelengths with the NIGHTSEA system requires three swaps (light head, barrier filter, and shield) and takes about 5 seconds.

Choice of excitation and emission wavelengths

• The NOZL systems are more versatile: virtually any wavelength combination can be implemented by installing the appropriate interference filters.
• The NIGHTSEA system currently offers 6 of the more common excitation/emission combinations.

Bulb warm-up/cool-down

• The LEDs in the SFA are instant on/off, with no warm-up or cool-down required.
• The HBO sources (high pressure mercury vapor arc-discharge lamps) in most NOZL systems require several minutes warm-up and cool-down.

Portability for demonstration and outreach

• The SFA system is highly portable and can be readily transported for demonstration and outreach.
• The NOZL systems are larger and less amenable to relocation.

Observe fluorescence through the viewing shield

• The SFA uses interchangeable shields matched to each excitation wavelength.
• The NOZL systems typically have a single shield, not matched to particular wavelengths.

Expense

• The SFA system is significantly less expensive than the NOZL systems, by a factor of 10 to 20 or more depending on options selected for each.

Customer comments on SFA performance

The systems from NOZL are generally terrific and we are sure each of the companies can provide nice things that people say about them. Below are some things that NIGHTSEA users have said about the SFA.

For bright specimens they completely replace the need for a traditional fluorescence built into the stereomicroscope. We had some issue detecting red fluorescent proteins with some of our weaker transgenic zebrafish lines, but by shining two LED lights onto the same embryo, most of our weakest GFP and mCherry transgenic lines can now be detected. One light head was fine for reasonably bright specimens, and with two lights, the fluorescence is nearly the same as traditional stereomicroscopes priced $15-20K. [zebrafish researcher]

The fluorescence adapters allowed easy viewing of the GFP expression at a magnification needed to view the entire root directly through the petri dishes in which the plants were grown. The fluorescent signal was on par with (if not brighter than) the reporter fluorescence observed at higher magnifications using confocal imaging. [Arabidopsis researcher]

The strength of the signal is excellent allowing any individual to identify tissues expressing GFP at multiple magnifications. [Drosophila researcher]

The NIGHTSEA works perfectly for the purpose of animal sorting, is very easy to use, student friendly, very portable and a 10th of the cost of a fluorescent dissecting scope. [Drosophila and C. elegans researcher]

I would like to emphasize that I am delighted with the power of the royal blue illuminator. It is nearly as good as the epifluorescence and much more convenient! I will be recommending it to my colleagues for classes. [zebrafish researcher]