Microscopy Matters

Insights from the Advanced Imaging Center at Janelia

What makes a successful proposal to the AIC?

One of the most common questions we receive at the AIC is: what exactly constitutes a strong proposal – what does the committee look for? What is the recipe for success?

In this blog, we will explore what makes a proposal stand out favorably amongst its competitors in each review process, and what most of the successful investigators have done to get it right. As outlined on the AIC website, the proposals will be reviewed on a two-tier basis. Tier 1 is the technical review by the AIC team, and Tier 2 is the scientific merit review. Let’s explore in greater details what each committee looks for during the review process.

Tier 1

The purpose of the Technical Review is straightforward. We want to address (i) whether it is feasible to perform the proposed experiments at the AIC, (ii) if the applicants have exhausted the capabilities of commercially available instruments and therefore have justifiable needs to use the AIC instruments, (iii) if the applicant makes the optimal instrument choice, and (iv) if there are quantitative elements in the experimental design. In Tier 1 review, only a pass/fail will be assigned.

Feasibility and Justification

Based on the criteria listed above, a successful proposal would state concisely and precisely the central questions to be addressed during the short stay at Janelia, supported by strong preliminary data showing that none of the commercially-available instruments (laser scanning confocal, spinning disk confocal, SIM, STORM, STED, EM, etc.) that they have tried can get to the gist of the problem. An example of a description that addresses criteria (i) and (ii) would be:

We aim to dissect how our motor protein of interest mediates vesicular trafficking within a specimen thickness of 300-400 microns. Currently, both spinning disk confocal and two-photon microscopes have failed to combine the simultaneous need for speed, reduced photostress and penetration depth (See figure N). Specifically, we seek to acquire optical sections at 1 stack per second, at a depth of 300-400 microns, for 5 min. Our vesicles are 200nm in size and distributed sparsely enough that we can easily resolve them using a standard confocal microscope. We will employ GFP and TdTomato to label our vesicles and the motor protein. However, using conventional methods, we are unable to image more than 30 stacks due to photobleaching, or pass 100 microns in depth, due to limitations of commercially-available microscopes.

Note how the experimental plan clearly delineates the penetration depth, image acquisition speed, imaging duration, the required resolution, and the fluorescent probes used in the sample. More importantly, they justify the need for AIC instruments by showing preliminary data obtained on commercial instruments (presented in Figure N in their proposal). They even give the AIC a hint of how photostable their fluorescently labeled proteins are. This is a well-advised proposal, indicating that they have exhausted all available options to earnestly address the imaging depth and speed issues, and they are facing a real technical hurdle that justifies the involvement of the AIC.

Note that it is not the mission of the AIC to address the paucity of commercial instrument at the applicant’s institution. It is also not the purpose of the AIC program to help you defray the cost of long duration imaging simply because the hourly recharge rate is prohibitive at your institution. It is not advisable to use these factors as justification of needs.

It is quite common to see applications with preliminary data that are full of immunoblots, bar charts, and a simple widefield epifluorescence image. In general, the preliminary data should be supportive of your justification to use the AIC instruments; it should not be treated as a justification for Janelia to fund your entire research project. So do avoid writing a five-year research plan. While it is advisable to include data that will highlight scientific impact, one must include preliminary data (or cite known technical limitation) to show that one has considered other imaging modalities but failed to obtain the results to address the central scientific question. Showing a simple epifluorescence image is usually insufficient. Likewise, long duration experiments (such as those that last for many hours) usually do not require high-speed imaging, and thus do not justify the use for some of our high-speed imaging systems. The justification of need is often the weakest aspect of most failed applications because most applicants justify their need solely by scientific impact, and not why they technically need the AIC instruments. Both aspects are equally important to be successful.

Alternatively, another acceptable, but less common, justification to try the AIC instruments comes from those who have extensive engineering support and who are planning on cloning the equipment here for their own lab or institution. However, the applicants still need to justify why they would need the microscopes at the AIC, as mentioned before.

Another common oversight is poorly conceived, or the complete omission of, labeling strategies. For example, while a few instruments at the AIC are tailored for single molecule imaging, it does not mean the microscopes can see single molecules without proper consideration of background noise and well strategized labeling techniques such as using photoconvertible fluorescent proteins. If the labeled protein is over-expressed or diffusive in the cells, single molecule imaging is not possible. Consult with the AIC team early before you start writing your proposal.

The AIC team can be best reached at aic@janelia.hhmi.org

If your lab is not specialized in advanced optical imaging techniques, then the best resource to help you address these issues is, first and foremost, the microscopy core facility of your institution. The core personnel should be able to point out the exact technical limitation of commercially available instruments (whether or not you have them in the institution) to you, and how your experiments may benefit from the AIC. They will also be able to help you generate the preliminary data. Ironically, it is often the case that in the process of generating the preliminary data, many prospective applicants obtain the answers that they need using conventional instruments, thus obviating the need to approach the AIC.

Instrument choice

The technical details of the AIC instruments are listed on our website (http://janelia.org/aic). We understand that the technical information can be daunting for biologists whose primary expertise is not in microscopy. This is where the AIC team can help. Start your proposal process early, send us a draft of your proposal, and stay communicative with the AIC team. We are here to steer you in the right direction once you have provided us detailed information such as that presented in the example above. There are times when we will recommend for you to try more than one instrument, and you are allowed to request more than one instrument for your visit – as long as you have good technical justification. And we can help. The AIC team also often asks the applicants to scale back their ambitious experimental design when it exceeds practical limitations. For example, it is not feasible to image thousands of images per minute for hours in duration. Your biological system does not have that photon budget, and the sample will bleach prematurely. However, you need to give us the opportunity to provide you such feedback. So start early.

Quantitative elements

“We want to look at microtubule dynamics”, proposed an applicant. “We want to see how the cells migrate”, proclaimed another. These are examples of proposals devoid of quantitative elements. Qualitative proposals with ill-defined plans to quantify the data usually score poorly, especially in the 2nd Tier, as they indicate that the applicants have not considered carefully what they plan to do with the data.

Think about precise measurements you would like to perform. For example, if you are interested in vesicular trafficking, then explore various ways of measuring movement: average velocity, instantaneous velocity, angles, persistent length, frequency of saltatory movement, etc. The AIC provides extensive data analysis support. It is perfectly acceptable to tell us what you want to measure, but ask us for help in measuring it because advanced digital image analysis is not your field of expertise. It is, however, not the responsibility of the AIC to tell you what to measure. While the AIC team will not fail your proposal due to lack of quantitative analysis in the technical review, our concern in this aspect will be summarized in the report submitted to the Scientific Review Committee.

Tier 2

The Scientific Review Committee consists of experts in cell biology, molecular biology, instrument design, imaging probe development, and imaging applications; and includes both Janelia and external representatives. The committee specifically looks for the strength of the research strategy, and the potential scientific impact.

Research Strategy

The second tier committee will evaluate if the scientific rationale presented in the proposal supports the project and whether the applicant gives sufficient consideration to potential problems and addresses alternative approaches. More importantly, we specifically look for detailed description by the applicants on how they plan to use the information generated by the AIC instruments. Common concerns from the committee include: (i) lack of indication that the applicant knows exactly what to do with the data; (ii) the applicant has no way of gaining mechanistic insights using this approach; (iii) lack of statistical analysis (because the applicants just want to look at a few specimens and “see what happens”). Taken together, these comments further reinforce the importance of quantitative methods presented in the proposal. So highlight in detail how measurements will be taken and analyzed, and include a description of all necessary controls.


The AIC instruments are powerful tools, and we want to see them put to the best use with projects highly focused on gaining mechanistic insights into biological processes. It would be considered poor justification to use the AIC if the committee perceives an application merely as an attempt to obtain “a prettier picture”. It is therefore important to elaborate why deciphering the biological mechanism in your project is significant, and to articulate how gaining this novel knowledge will affect the field of study. It will also be helpful to describe how you plan to leverage the newly gained knowledge for future experiments. This will address the common concern about the lack of clarity of how the data can be helpful.

Other considerations

The AIC does not put a cap on the length of stay at Janelia. However, the duration must be well justified by experimental needs. The average visitation is 1.5-2 weeks. Please note that the AIC will not schedule more than one group of investigators on each instrument at any given time. So take into account the rate of data output from the exclusive availability of the scope when you budget for time. Consult with the AIC team for recommended length of visitation if you are not sure.

The committee does not differentiate between principal investigators who are at the professor, postdoc or graduate student levels. All proposals are evaluated on their own scientific merit. Our past experience was that proposals submitted by professor level or postdoctoral level principal investigators received almost comparable rate of success.

In summary, leverage the availability of technical consultation with the AIC team to the fullest and start the proposal process early. Make sure you justify from a technical standpoint why you need to use the AIC instruments, and provide detailed strategy of quantitative image analysis.

Contact us early
Open dialog
Note clearly what to analyze
Specify experimental parameters
Understand AIC scopes
Labeling strategy
Tackle mechanistic insights
Articulate scientific impact
Include controls
Consult us!


Teng-Leong Chew serves on both the Technical and Scientific Committees.

Author: Teng-Leong Chew, Ph.D.

Center Director

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