The nematode is only about 1mm, the body is transparent, swimming and fast, it seems to be the "easiest to observe" experimental material, but in fact it is pitted everywhere: just right, the scorching worm swam away, the state of the sample deteriorated after a long time of light, and half of the photos taken were virtual.
This article breaks down the practical process of observation of live nematodes into 3 steps: sample preparation → observation skills → imaging records, and gives a checklist of common problems. Equipment selection suggestions are attached at the end of the article.
Understand the pain points before you know why you're doing it every step of the way:
The nematodes are picked from a petri dish, and the action should be light, avoiding excessive media.
It is recommended to use a shallow bottom culture dish or slide + agar pad for observation vessels, the thinner the liquid layer, the easier it is to focus.
Commonly used M9 buffer to maintain osmotic pressure, the amount should not be more, just a thin layer.
If the liquid layer is too thick, it will cause the insect body to move upstream and downstream, and it will scorch frequently.
Platinum silk worm picks or eyelash pins, which are picked from the back of the worm body with a light strap and do not poke into the head.
Clean the needles after each pick to avoid cross-contamination.
Anesthesia: Low concentration of L-imidazole can inhibit movement for a short time, which is easy to take photos for measurement; pay attention to control the concentration and time, and transfer in time after observation.
Cooling: Low temperature can reduce metabolism and movement speed, suitable for short-term observation, but do not leave the sample on the cold table for a long time.
If it is only rapid screening, it can also limit the activity of insects by reducing the liquid layer, reducing the interference with the sample.
First, scan the entire visual field to locate the worm body with a low-power, large field of view, and then gradually increase the size of the head, pharynx, and tail structure. Avoid going up high, so the field of view is too small and the worm quickly swims out of the picture.
The insect body is located in a thin liquid layer, and the coarse tuning is easy to cross the focal plane. First use rough adjustment to roughly align the liquid level, and then use fine adjustment to find the outline of the insect body.
High light stresses, curls, and even kills nematodes. Using soft transmitted light, start with low brightness and tune up gradually, stop adding light as soon as clearness is observed. Observe for a long time to turn off the light intermittently.
Improved contrast doesn't have to be light: a white or light gray background can be placed underneath the dish, or the silhouette can be enhanced with lateral illumination, which is gentler than the slightest increase in brightness.
This is the most overlooked point. The thick → insect body of the liquid layer → frequently runs scorched in three-dimensional space. The liquid layer is controlled to the extent that the insect body has just been eliminated, and the insect body is basically confined to the same focal plane.
It is recommended to control the single continuous observation for a short time, and let the sample rest in the middle; when long-term recording is required, video recording is preferred instead of continuous visualization to reduce the total duration of light.
Nematode movement is fast, and it is easy to miss the key action in continuous shooting. Record the complete process with video, and pick out pictures frame by frame afterwards, which is more reliable than on-site capture.
Before measuring the length and width of the body with image measurement software, first use a ruler or a known size to calibrate, otherwise the measurement data at different magnifications will not be comparable.
In vivo nematode observation has completely different equipment requirements than looking at fixed sections:
Large depth of field - the insect body has a thickness in the liquid layer, and if the depth of field is too shallow, it will be half empty and half real;
Continuous zooming - first look for insects at low zooms, then look at the details at high zooms, without changing the objective lens in the middle;
Soft transmission illumination - no damage to the sample, suitable for long-term living body observation;
trinoculars + camera - can take photos and videos at the same time, which is convenient for recording and measurement;
Large operating space - Petri dishes can be placed directly on it, making it easy to pick insects and dissect them.
Ordinary biological microscopes (upright, high power, strong light) are not suitable for these tasks; stereomicroscopes are the main equipment for daily observation of nematodes.
ST118Z is a trinocular stereoscopic video microscope designed for nematode observation, biological sample dissection, and live specimen observation:
Continuous zoom optical lens with large depth of field and wide field of view: The insect body does not easily run out of the focal plane during swimming, and low-power scanning and high-power observation can smoothly transition;
trinocular interface can be connected to the camera, synchronous computer imaging: visual and screen at the same time, suitable for students with experiments or multi-person observation;
The transmitted light source is soft and does not hurt the sample: suitable for long-term observation of living nematodes;
Support photography, video recording and image measurement: observe the length and width of the direct measurement body, eliminating the trouble of replacing the equipment;
It is suitable for university laboratories, biological research, agricultural plant protection and other scenarios.
For laboratories that require batch screening, instructional demonstrations, or long-term documentation, this combination of “stereoscopic + video” is much more efficient than a simple visual microscope.
Q1: What microscope is used to observe live nematodes? A: Stereoscopic microscopes (large depth of field, long working distance, and direct placement of petri dishes) are preferred for daily picking, sorting, and in vivo observation; biological microscopes with higher magnifications are used when cell-level details need to be viewed. It is recommended to choose a trinocular stereo microscope with imaging needs, which can be connected to the camera at the same time.
Q2: What if the nematode keeps swimming and can't see clearly? A: Priority is given to thinning the liquid layer, followed by short-term cooling or low-concentration anesthesia, and finally increasing the shutter speed with video recording. It is not recommended to use a high concentration of anesthesia as soon as it comes up, which will affect the state of the sample.
Q3: What causes nematodes to die easily when observed? A: Most of them are caused by excessive light or evaporation of the liquid layer. Reducing the brightness, shortening the single observation time, and replenishing the liquid in time can basically solve the problem.
Q4: The nematode body length needs to be measured, what should the device meet? A: It is necessary to be able to connect the camera and accompany the image measurement software, and support calibration. trinocular video microscope + measuring software to complete, no need to purchase additional measuring instruments.
Q5: Can plant nematodes (agricultural samples) also be used? A: Yes. The in vivo observation and morphological identification after the separation of plant nematodes are also suitable for stereoscopic microscopy, combined with transmission illumination to observe structures such as the stylus and tail.
The key to live nematode observation is not "the higher the magnification, the better", but: thin liquid layer, soft illumination, low magnification first, high magnification later, video instead of snapping, calibration before measurement.
If the device is selected correctly, these techniques will be easy to land on the ground. The Yikamo ST118Z nematode stereoscopic video microscope has been specifically designed for in vivo observation - continuous zoom, large depth of field, soft transmitted light, trinocular connectable camera, suitable for daily nematode observation and recording in university laboratories and scientific research units.
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