Olympus CKX41+trinocular observation tube split 50% light for camera, acceptable for taking fluorescence pics?

[tocharian]

I'm a researcher in China. I intended to procure an entry-level inverted fluorescence microscope for research purpose. I planned to use this to observe fluorescent bacteria cells (1-2 microns), and take pics with my DSLR. In the future, I want the system to generate 3-D view of microbial biofilm (thickness tens of microns), if possible (heard that this can be done by manual z-moving and stack the images).

I ordered Olympus CKX53 (w. uplfln40x, uplfln100x and cheap 4x, 10x objectives)+fluorescence accessories from a company. But the company failed to deliver the microscope after 6 months, and is now offering to supply CKX41 instead. Of course I have the option to cancel the order, but if CKX41 is not really unsuitable for my purpose, I’d rather not to do so. 1. Prices from official local Olympus suppliers are 30-50% higher than these listed in the North America; and this company offered much more attractive prices since their source of supply is unofficial (but also unstable). 2. I already paid 60% of the agreed amount in June, and received the fluorescence accessories. It takes time/energy to fight the company, get money back, and find new supplier,,, while I want to start my research as soon.

Now this company is offering CKX41 with trinocular observation tube “U-CTR30-2-2”. First problem with the accessory is that, it has FN20, not FN22 of CKS53’s. A seemingly more serious problem, is that, the tube does not come with light path switch, but split light 50/50 between eyepieces and camera! Means camera always receives only 50% of light. There is “U-TR30-2-2” with FN22 and light path switch. But its list price in America is like $1,700, and the company says never head of it. My choice (if stays with the company) is either to accept this configuration or wait for CKS53 (not sure if can get it after another 6 months).

What I want to ask here is:
1. How is CKX41 compared to CKX53? Is the body (except for the observation tube) equally good for my purpose?
2. I expect that there is an effect if only 50% of light reaches the camera. But, in practice, how severe the effect will be, Considering my purpose (fluorescence observation needed) and other configurations (objectives, DSLR…)?

Thanks.

[billbillt]

Hi,

I would get quotes from other suppliers with different brands... Olympus is very good, but they are not the only one...

BillT

[tocharian]

Hi,

I would get quotes from other suppliers with different brands... Olympus is very good, but they are not the only one...

BillT

Thanks, I wanted to stay with mainstream brands, although my budget is limited. Olympus is the cheapest among the 4 brands, the other brands also have higher price in China than in America, Japan.

Another problem is that, I already own uplfln40x, uplfln100x (they are not from that company). If I get another brand body, I need to figure out what to do with these objectives, not many people need them, and they are not cheap.

Actually, the company offered to supply an entry-evel inverted Leica, but I turned that suggestion down, due to the aforementioned reason.

[Hobbyst46]

Although I am not familiar with the 41 model and its difference from the 53 model, I think I can input some relevant information.

First, recording images of individual 1-2 micron fluorescent bacteria with A CAMERA is not easy. The light level is initially low. What is the numerical aperture of your 40X lens? it must be a high NA immersion objective for the purpose.
Olympus sells dedicated cameras, for example the sensitive DP-73 (or similar). You need to buy it together with an appropriate adapter (with a 0.5X or so relay lens) to have a decent FOV with the microscope and that camera. I am aware that these cameras are expensive. But I am not sure that a DSLR is sufficiently sensitive for your specific target.

Second, I do not think that the 50/50 (eyepiece/camera) configuration is very important. A factor of 2 in the intensity of the emitted light is not critical.
Likewise, for your type of specimens, I think that FN20 vs FN 22 or higher is not so important.

Third, I believe that the quality of the results you expect depends much on the fluorescence accessories. What is your illumination source? how efficient are the cube filter and mirror components? these should be of the highest quality, since the challenge is signal to noise. That is, the separation of the fluorescence from the background. The latter is determined by the excitation spillover and from ambient light.

Fourth, the strongest illumination source that I know is still a 100-150W high pressure mercury lamp or 75W xenon lamp. There are LED sources, like the PE-100 or 300, but I suspect that their intensity is lower. Can you inquire about this with your supplier? LEDs have advantages ,but for your specific purpose, I would try and verify the intensity.

In addition, I recommend that you prepare a well-darkened work area for the microscope, or even placing the microscope within a light-blocking box, such that ambient light around the stage and below it is negligible.

I somehow believe that the above points/issues will determine the results more than the differences between models 41 and 53.

As for Z-stacking - would'nt is be better to do it with a confocal microscope, rather with camera-based microscopy?

[tocharian]

Although I am not familiar with the 41 model and its difference from the 53 model, I think I can input some relevant information.

I understood from your recommendation that, the signal/noise ratio and resolution are more important than the absolute brightness. Also, am I right that, illumination and the perceived brightness has a logarithm relationship, not linear?

The fluorescence accessories are not from Olympus but all made in China. The light source is indeed LED, and here is a photo http://www.sypower.com.tw/pcontent.php?pid=121 The filter sets are assembled by the company, claimed to be made of imported filter pieces. Considering the price, I cannot expect them to be the highest quality.

The only highly configured part of my system is probably the objective. Uplfln is fluorescence lens typically used for more expensive research grade microscopes. Still, the NA is not much larger than the cheap objectives. 40x (dry) has NA 0.75 and 100x (immersion) has NA 1.3.

My original plan was to use my personal DSLR as the camera. The company will provide me a converter as “gift” but I didn’t ask about details. I cannot afford advanced microscope cameras (e.g. w. cooling), but may be able to afford pre-owned mid-low class Olympus cameras. I thought that DSLR has larger pixel size than ordinary cameras, and should be more suitable for capturing low-light? Am I wrong?

Actually, I don’t expect to identify the detailed structure of individual bacterial cells. Even with the 100x objective, there are hundreds of cells in the FoV. I wonder, will my system possibly generate images quality/resolution close to this? Any other recommendations that I can improve my setup without much further monetary investment?

Thanks.

[Hobbyst46]

Although I am not familiar with the 41 model and its difference from the 53 model, I think I can input some relevant information.

I understood from your recommendation that, the signal/noise ratio and resolution are more important than the absolute brightness. Also, am I right that, illumination and the perceived brightness has a logarithm relationship, not linear?

I will try to answer from experience and knowledge as best as I can. (1) Signal to noise and resolution are mutually contradicting, but for fluorescence, you want the optimum of both. (2) A high signal to noise is more important than "brightness". For example, a very bright image will have many over-exposed ("saturated") peaks in the image. (3) The "illumination" in this case is actually excitation. The intensity of fluorescence is proportional to the intensity of the excitation, so you want intense excitation, but that in turn might cause photo-bleaching as well as photo-damage. The latter might be unimportant with bacteria - I do not know. (4) I do not know if and how a logarithmic relationship plays a part there.

The fluorescence accessories are not from Olympus but all made in China. The light source is indeed LED, and here is a photo http://www.sypower.com.tw/pcontent.php?pid=121 The filter sets are assembled by the company, claimed to be made of imported filter pieces. Considering the price, I cannot expect them to be the highest quality.

Since the light source is LED, it is a relatively narrow wavelength range. Often a LED alleviates the need of excitation filter, although, for small Stokes shifts, I would still prefer an excitation filter. Experience will then show you if the LED light is of sufficient intensity. About the "quality" of the filters and dichroic mirror: I would judge it from the transmittance spectra. Contact the company and ask for the names of the makers of the optical components, the models, and the spectra. BTW, transmittance spectra of components from the top-level optical mfg. are available on the web (e. g. Omega, Chroma, Edmund, Newport etc). Incidentally, What diameter are the filters and what was the price?

The only highly configured part of my system is probably the objective. Uplfln is fluorescence lens typically used for more expensive research grade microscopes. Still, the NA is not much larger than the cheap objectives. 40x (dry) has NA 0.75 and 100x (immersion) has NA 1.3.

The objective is the single most important component of the setup, since it serves two functions: as objective and condenser!. The signal to noise is very strongly dependent on the NA (to the 4-power if I am not wrong!). From the posted photo of your LED I take it that your excitation wavelength is visible, not UV. Because high-end objectives are highly transparent to UV, so if your objective is of this kind, it will probably be more than adequate. Anyway, bear with me for saying that an NA of 0.75 is much too low for your application, unless you only need it to locate ROIs. But for the latter purpose, I would prefer a 10X-20X objective.

My original plan was to use my personal DSLR as the camera. The company will provide me a converter as “gift” but I didn’t ask about details. I cannot afford advanced microscope cameras (e.g. w. cooling), but may be able to afford pre-owned mid-low class Olympus cameras. I thought that DSLR has larger pixel size than ordinary cameras, and should be more suitable for capturing low-light? Am I wrong?

I can only say that, for your typically extremely low-light application, the most important feature of the camera is sensitivity. Outweighs any other feature.

Actually, I don’t expect to identify the detailed structure of individual bacterial cells. Even with the 100x objective, there are hundreds of cells in the FoV.

Agreed.

I wonder, will my system possibly generate images quality/resolution close to this?

Not with the 40X0.75 objective.

Any other recommendations that I can improve my setup without much further monetary investment?

If possible, take your best specimen/slide (best in terms of expected fluorescence!) to a fellow microscopist with access to an epi-fluorescence setup, record images, and compare the results with the optical features of the setup, it may well direct you to a solution.