In Vivo Imaging - Science method

What is the absorbance spectrum of the pigmentation? You could check if there is a wavelength where the pigmentation is transparent, but the neurons (or whatever you're interested in) absorb.

One big challenge would be the opacity of the tissue. To measure fluorescence, excitation light must reach the fluorescent substance, and the emitted light must reach the detector. In both cases, the tissue would absorb and scatter the light. You would only be able to measure fluorescence from the surface of the exposed tissue. The depth of light penetration increases with the wavelength, so the longer the excitation wavelength of the indicator the better.

A second big challenge would be the background fluorescence from the tissue.

A third challenge would be delivering the calcium-sensitive dye to the tissue in a living animal. I think microinjection has been used for nerve cells.

There is a high concentration of calcium in the extracellular fluid and a low concentration of calcium in the cytoplasm of cells, so any intravenously or intraperitoneally delivered indicator is probably just going to show you the extracellular calcium concentration.

When you threshold, don"t click "apply". That's all! So, after you split the channels, you go to Image>adjust>thereshold, and then Analyze>measure. Make sure you have gone to "set measurements" first, and selected the "linit to threshold option".

Manuel Gómez Thanks a lot for the articles. I am working on a literature survey actually and I am particularly looking for any research from the past 5 years that have used carbon allotropic nanomaterials for imaging viruses. Since 2016, I have found only 1-2 nanodiamond based virus imaging studies. I know that all these allotropes generally have fluorescent properties and they have been used for bioimaging a host of different targets. However, what I couldn't find is any recent progress on such materials within the past 5 yerars particularly for viruses. For example, I have tried looking for single virus tracking using these nanomaterials and the search always comes empty. By tracking, I am talking about in vivo-in vitro imaging of different things such as cells/ zebrafish embryos. mice using microscopic imaging methods such as bright field microscopy, etc. Although the papers you have refernced in your response are helpful to gain a better understanding of the nanomaterial's characteristics in the context of fluorescence, I could unfortunately not succeed in finding any particular research pertaining to viruses or justifying the absence of it

Sorry for the delay and thank you for this helpful input about stability!

The dosage and concentration can depend on a lot of factors like the route of injection (i.p, i.m, oral, etc), the buffer in which you want to dissolve.

Hi,

The same advice for using phase contrast MRI !

Best !

Steven Hou , please, could you share how you solved the re-growth of bone in your windows? I am experiencing the same problem...

Hi,

I wonder whether you have established the model successfully? I am now working on the same model as you described above and similar problem occur to me. I am curious whether you have worked it out?

Regards,

Dan

I think you might be confusing cancer cells with macrophages - yes cancer cells can deplete resources and accordingly stress tissues, but they do not "consume" cells like macrophages do.

Definitely not a hind limb of the mouse, but the MR image quality is too poor to tell the structures for sure.

Amplex red is not a good option for this case because the dye could be oxidized during the delivery and the oxidation reaction is out of control since the peroxidases are just abundant in vivo. In this case, a better option would be the genetically encoded protein sensors, but it takes quite some time to produce mice expressing either Hyper or roGFP sensors.

Br. J. Nutr. (1967), 21, 413

Interrelations of calcium, fluorine and vitamin D in bone metabolis

DEBORAH GASTER, E. HAVIVI, K. GUGGENHEIM

Bioluminescence depends on the ability of the emitted light to penetrate tissue, most animals are too large for this unless the area of interest is in the skin. This is the reason most BLI studies use mouse models as with a red-shifted luciferase and a high enough expression level you can detect signal from deeper tisues (Red light exhibits less scattering and is not as highly absorbed by hemoglobin as green/yellow wavelengths).

Thanks Ondrej

Dear Saleh, Islam and Amin, 

Thank you very much for your answers.

Hi Abhimanyu, thank you for your reply! Do you have any recommended ones to use for RFP?

Depending of available excitation laser channels (635, 680, 750, & 790 nm) you can select appropriate cell tracker on attached page. Take in mind period of tracking (cell generations) during selection of dye. It seems CellTracker™ Deep Red ( 630/650) will be suitable for relatively short tracking (days-weeks) and Qtracker® family (>650 nm) for long period.

Of course I've attached page for informational reasons and you can try to find same technology from other suppliers.

Best wishes!

Daniil

Thank you very much for your explanation and recommendations, they are very useful.

Regards

Natalia

Yes you can substitute provided you have enough facility in your lab to get or measure the labelled C in that particular tissue.

I've found that, depending on where you inject, if your needle is subcutaneous, you're likely in the fat pad. I recommend doing a few practice injections with PBS + trypan blue or hematoxylin. After injecting, sack the mouse, and make sure the dye stayed in the fat pad. You'll find that if you're in the right spot, you'll rarely miss!

It is not only the scattering (but it is the most important part), but also the absorbtion of blood that makes NIR more feasible for in vivo Imaging. On the other side you will get  absorption of water, if you are using longer wavelength above 1µm.

Here is the source of information on the uses of Alexa Fluor dyes:

I would also suggest to try the alfalfa free diet first.  The fluorescence lifetime of Cy5.5 is shorter than the sources of autofluorescence, but I guess that option is not possible if you are using an IVIS.  I have tried spectral unmixing, the autofluorescence has a broader excitation band than the Cy5.5.  However, at low concentrations of dye (similar to autofluorescence) I never really got it to work very well.

The other obvious thing is to try to place the Cy5.5 compounds in an area as far from the intestine signal as possible, and then shield the intestine.  If the excitation light does not reach the area of strong autofluorescence you can avoid a lot of signal 'leakage', just as you might shield the bladder and/or liver if there is accumulation here due to excretion routes.  Also try repositioning the animal and/or sheilding masks to reduce the unwanted signal.  Apologies if this suggestion is too 'obvious' and you may well have already tried this.

Could you post an example of the background?  And perhaps a sketch of the prep?  I don't understand where the interference might be happening.

We try to keep the power <40mW when imaging in the cortex 100-300 um below the surface.

For deeper imaging you can increase the power since more of the excitation light will be scattered out of the focal volume of your objective and you are less likely to damage the tissue.

You still need a bright fluorophore to get a signal from 500 um or deeper.  A good way to test may be labelling the vasculature with dextran conjuagted FITC or use a transgenic mouse line with the Thy1-YFP which expressing YFP in layer 5 neurons.  The cell bodies are bight and around 500 um deep.

Just try to use diluted red blood cell suspension.

Maybe this video might help.

Systemically circulating amidolytic thrombin activity, i.e. thrombin complexed to and transported by alpha2-macroglobulin can easily be measured in EDTA-Plasma using a fast chromogenic thrombin substrate and an incubation time of 1-2h (37°C).

Dear Mustafa,

One of the methods to be used is treating your TEA solution with ZnCl2 and run IR spectra (attached is a paper for conducting such experiment.

Raman spectra can be do the job.

Good luck,

Rafik

Have you tried using stereozoom microscopy? You may prepare contention device using wood and velcro. You can check the model, where you can get 10-40x magnification

HI Sophia

In our lab we used  the retroviral transduction with a pMig-GFP vector and I could see Marked T cells at least after 8 weeks (not sure for long term experiments)

Can't imagine in vivo imaging will help so much. You should do IHC for neuronal markers - which won't tell you if they are functional - just that they exhibit those markers. for functional studies, you might need vibratome sections for electrophysiology, or use minimacs separation for ex vivo functional assays (but not sure which one)

Synchrotron radiation technology （e.g. EXAFS） is a kind of preferable method to be used to trace metal ions in vivo.  

Perhaps,  the following paper will be helpful.

Smirnov, I. V., Kotch, F. W., Pickering, I. J., Davis, J. T., & Shafer, R. H. (2002). Pb EXAFS studies on DNA quadruplexes: identification of metal ion binding site. Biochemistry, 41(40), 12133-12139.

Good Luck!

then try

Dear Farzad:

I will be glad to be of any assistance. I have more than 10 years of experience in preclinical cancer therapeutics - animal studies. 

Thank you very much for informative review. MITF-M isoform exhibits the complexity in the differentiation, proliferation, cell cycle regulation, invasion and metastasis. It has been quite controversial to the degree of which Dct promoter contributes to the melanocyte-linegage in a specific manner. Indeed, Dct is one of the target genes for MITF-M, and furthermore, given that Dct-H2BGFP-Tg mice is the main tool to identify the melanocytic cell-lineage as well as Tuj1+ peripheral nerves, this review might be frustrating for my current PI.

I think you may need to go to a different pressure-sensing technology, i.e., one that senses pressure closer to the tip of the implanted catheter. I am attaching a paper that describes one such unit that seems to work well in mice. Here is also the link for the company (Data Sciences International) that commercialized the unit (I think). I have never used these units so buyer beware.

Agree with Grazvydas.  Not sure why you are adding water to your dye.  The low ionic content of water especially pure water in a lab may well not allow full solubilization.  Try dissolving in PBS.  As a good technique always spin antibody solutions before use.

I tested the effect of four concentrations of magnesium chloride on daphnia mobility.

0.25M killed the daphnia in 20 minutes. 0.03M had very little effect over a 95 minutes observation period. 0.13M made daphnia immobile after 15 minutes, keeping them alive for about 80 minutes. 0.06M gradually slowed down their swimming and finally immobilised the daphnia after 95 minutes.

Therefore, 0.13M MgCl₂ would work fine although the impact on daphnia physiology is probably significant.

Thank you for the suggestion!

While ICP-MS is a gold standard for it, one can also use AAS (atomic absorption spectroscopy) for this purpose.

Hi

I would suggest you that in 12 well plate prepare Poly-L-Lysine coated glass, put 2 million cells in 1 ml of  either medium or PBS, centrifuge 15 mints at 1000 rpm. I had done several times. For sure my cell line isn't MOLM13-cells, good luck!

Hi,

as mentioned above we administer  a mix of ketamine/xylazine or gaseous isoflurane in pure oxygen, too. These two anesthetics are a good choice for terminal experiments combined with a craniotomy (leaving the dura intact) for direct access to the brain tissue for patching. We keep the eyes protected with an eye creame, monitor the breathing and stabilize the body temperature with a heating plate.

For awake measurements you need to train the animals to adjust to the head fixation for instance before you start your experiments.

There is of course the possiblity to use an antagonizable 3 component anesthesia  ( i.p. injection) composed of Fentanyl 0,05mg/kg Bodyweight (BW), Midazolam 5,0 mg/kg BW and Medetomidin 0,5 mg/kg BW)  and an antidot (Naloxon 1,2mg/kg BW, Flumazenil 0,5mg/kg BW, Atipamezol 2,5mg/kg BW), to wake up the animals within 2-5 minutes after s.c. administration. This is extremely helpful if the animals need to wake up after surgery, i.e. for stereotatic viral injections to stain target cells or for chronic window surgery for later repetetive imaging sessions.

I hope you are aware that you need a permission first  from your local  "Regierungspräsidium"  to do such experiments!  

Good luck for your experiments.

Another interesting point is that both tumor microenvironments and stem cell niches tend to be hypoxic. Depending on the metabolic rate of the cells this can lead to more or less ROS. A stem cell niche might be preserving itself from ROS during quiescence, conversely tumor stem cells might be driven to death while dividing in a hypoxic environment created by antiangiogenic drugs. It seems important to gauge the level of hypoxia, metabolic rate, and ROS buildup of a given niche or tumor model to get a real sense of what is going on.

By combining an environmental scanning electron microscope (ESEM), a gaseous analytical detector (GAS) and a peltier cooling stage, this combination technique provides near-instantaneous nanoscale characterization of interactions between individual water droplets and AgNPs. The attached publication could be helpful to gain an insight.

Are you making intracellular, extracellular or patch clamp recordings? What is you preparation?

For all you need a Faraday cage and to ground  your electrodes and any potential source of electrical noise - preferably to a single ground point. 50Hz noise (at least in the UK) is normally due to a power supply so it probably needs to be grounded, Good grounding may eliminate this noise, if persistent using a HumBug to eliminate the noise is better than filtering in your software at 50Hz - this is less than ideal as you may miss some of the signal you want and should only be you lest resort.

Another possibility is the photon imager from Biospace labs in France. It detects both bioluminescense and fluorescence in the living animal in vivo and has the temporal resolution you are looking for.

Hi Claus,

Actually my greatest experience is with awake rats using PowerLab for invasive measurement. The use of CODA is recent and only using awake mice. However, if you have the CODA system, I believe isoflurane is not bad for cardiovascular measurements.

Marli

You can go down to nm resolution with super-resolution microscopy using standard fluorescent dyes in various existing techniques such as fPALM, SSIM, STED etc. However this is highly limited how deep you can go

Thank you for your question!

There are numerous challenges along the development path which must be resolved for each new clinical situation and hardware configuration. For instance, the scalp and skull temperatures will change as well. However, we're developing a multi-frequency radiometer that can distinguish temperatures at different depths and thus overcome such hurdles. In any case, we have demonstrated that a precision and “accuracy” of 0.4 degC relative to the calibration point is possible in vivo when the radiometry system is carefully setup and calibrated for the specific clinical measurement configuration.

I and Paul Stuffer discussed your question further, and here is a more complete answer in case you’re interested:

Remember that MW radiometry is best for relative temperature readings and is dependent on calibration to one or more known temperature references.

The projected accuracy and resolution of microwave radiometry in vivo is highly dependent on specifics of the clinical application as well as radiometer hardware and software configurations.

For instance, accuracy of one large volume weighted average measurement of deep tissue temperature can be very precise and accurate, but that “accuracy” is essentially impossible to prove experimentally since any other measurement approach would measure a different volume. Certainly no single or multipoint implanted sensor can come close to characterizing the same volume-average tissue measurement, because the radiometric measurement is weighted by the distribution of receive antenna sensitivity defined by the radiation (=receive) pattern of the antenna integrated over the measurement frequency band and further weighted by the non-uniform receiver gain of the MW amplification chain (across radiometer frequency band) AND specific impedance coupling of antenna to tissue volume. Even true 3D MR thermal imaging would read a different volume average temperature from radiometry, since MRTI will weight the relative contributions of tissue temperatures within the ROI differently from the wideband radiometer receive antenna. The MRTI and MW radiometer volume average measurements might read the same if the volume being sensed was entirely uniformly equal temperature, but this is a rather non-interesting clinical case (i.e. dead patient). In that case a simple thermocouple inside the uniform temperature volume also correlates accurately with either MR or MW Rad temperatures. That is how we do laboratory phantom calibrations and tests of MW Rad temperature “accuracy”.

Recall that the radiometer electronics gives one received power measurement for one broadband radiometric reading. To produce an “accurate” radiometric equivalent tissue temperature (i.e. predicted tissue temperature), we perform extensive EM simulations of antenna radiation patterns into tissue at various frequencies across the radiation receive band of the radiometer electronics, and blend them together into an approximate average weighting function for that broadband antenna sensor. So the mathematics of the temperature conversion algorithm calculates an equivalent average temperature of a volume of tissue weighted by the distribution of received energy over the band from the antenna. If you have accurate a priori information about actual tissue temperature (preferably from a similar volume as you are trying to characterize) then you can “calibrate” your received power reading to translate to that specific volume average temperature. Then the radiometer can read this temperature and even changes in that temperature quite accurately (better than 0.4 degC) - as long as the following factors do not change:

i) impedance coupling of antenna to that tissue volume (so it is essential to maintain a stable connection of antenna, or somehow correct for any changes - though any correction would likely put a hit on accuracy. Best to keep antenna connection stable),

ii) outside EMI pickup (so shield EMI from rad sensor and average over longer time periods (10 s or preferably 30 s or more) to average out noise spikes)

iii) amplifier gain (this is systematic and can be calibrated out – we have already done this in the 0.4 degC accuracy demonstration over multiple hour experiment)

iv) temperature of the radiometer electronics and antenna itself (this can be calibrated out by thermistor temperature sensors on antenna and electronics box, and we have done this also as it is critical for long term stability)

Certainly there are foreseeable additional hurdles but we believe that when used carefully in appropriately controlled clinical applications, we can deliver “accuracy” of our calculated weighted average tissue volume temperature measurement on the order of 0.4 degC – even for long times of many hours. We will be moving soon to multiple frequency band, multiple antenna, and correlation mode radiometer systems that provide additional readings of power (temperature) from different and overlapping sense volumes. These multiple readings, from overlapping tissue regions, can be used to distinguish: a) temperature-depth profiles, b) calibrate and correct spurious temperature readings, c) improve overall precision, accuracy and spatial resolution of radiometric temperature predictions.

An additional comment: If knocking down your GOI negatively affects cell proliferation, viability, etc.; or you wish to knock the GOI down during development in a spatial and temporal manner; you may wish to use an inducible shRNA expression system. There are different types of systems. One is the tetracycline-inducible shRNA expression system [ref: Nature Protocols 2, 3257 - 3269 (2007)].

Hi Daniel,

you may try to write to Paulo Magalhaes from prof. Pozzan's lab in Padua Uni/Italy

maybe you can try directly thorugh their lab's page http://www.pozzanlab.org/group.php.

As I can recall correctly, Paulo was working on some pH-sensitiv GFP mutants. He is a very nice guy, and if it is possible he will help you.

Good luck

Roman

Was there a particular reason for delivering the luciferin retro-orbitally? Ive never had problems but have never delivered by that route. First things that come to mind are prolonged anaesthetic at too deep a level or luciferin being too cold when delivered.

Optimized protocol is stated in supplementary material and methods of paper pubmed-id: 22884313. Good luck!

Yes -- but difficult.

Dear Marca,

for sure you did it already, but just check the in vivo work by Arthur Konnerths group in Munich. You will find a lot of excellent studies using OGB1 in vivo.

Kind regards

http://www.ncbi.nlm.nih.gov/pubmed?cmd=search&db=pubmed&orig_db=pubmed&term=Konnerth+A.

This is difficult. My only suggestion is the inverter device from lsm technologies (http://www.lsmtech.com/). It will take some engineering for the mouse mount but should be workable.

I read through the other posts and I see that you wanted to do these on bees. That obviously rules out my answer. Sorry for the confusion.

My previous group (lead by Prof. Martin Kerschensteiner) has just published a Nature Protocols paper that deals with ways of labelling different cell types/structures in vivo in the mouse. It should be out next month.

In the meantime, my suggestion for nuclear labelling is Nuclear ID Red, by Enzo Life Sciences (cat. no. ENZ-52406). I have used it in mouse spinal cord without even removing the dura and it works really well.

SR101 can be tricky, but I think its worth a shot, though admittedly I only have mammalian experience. Perhaps support it with fluo-4/colocalization during in vivo imaging or just do some good old fashioned IHC looking at SR101/GFAP colocalization after your experiment.