Cycles of circadian illuminance are sufficient to entrain and maintain circadian locomotor rhythms in Drosophila

Light at night disrupts the circadian clock and causes serious health problems in the modern world. Here, we show that newly developed four-package light-emitting diodes (LEDs) can provide harmless lighting at night. To quantify the effects of light on the circadian clock, we employed the concept of circadian illuminance (CIL). CIL represents the amount of light weighted toward the wavelengths to which the circadian clock is most sensitive, whereas visual illuminance (VIL) represents the total amount of visible light. Exposure to 12 h:12 h cycles of white LED light with high and low CIL values but a constant VIL value (conditions hereafter referred to as CH/CL) can entrain behavioral and molecular circadian rhythms in flies. Moreover, flies re-entrain to phase shift in the CH/CL cycle. Core-clock proteins are required for the rhythmic behaviors seen with this LED lighting scheme. Taken together, this study provides a guide for designing healthful white LED lights for use at night, and proposes the use of the CIL value for estimating the harmful effects of any light source on organismal health.

The LER is a parameter explaining theoretically how bright the radiation of the emission spectrum is perceived by the average human eye. The LER was calculated with photopic spectral luminous efficacy function (V(λ)) and the SPDs of light source (S(λ)) (See Eq. S1). The maximum value of the LER is 683 lm/W with SPD of 555 nm green monochromatic light.
Similarly, the CER is a parameter explaining theoretically how circadianly (non-visually) bright the radiation of emission spectrum is perceived by the average human eye. The CER was calculated with circadian spectral luminous efficacy function (C(λ)) and the SPDs of light source (S(λ)) (See Eq. S2). The maximum value of the CER is 683 blm/W with SPD of 460 nm blue monochromatic light.

SI 1-2. Circadian action factor (CAF)
The ratio of CER to LER indicates the CAF (Eq. S3). This value means the circadian response (biological action) per unit of vision response.

CAF (blm lm
The correlated color temperature and color, especially blue, are mostly effect on the circadian performance. The daylight, CCT of 6,500 K, has CAF value around 1.00. High CCT lamp (bluish white) has high CAF value and low CCT lamp (reddish white) has low CAF value.

SI 1-3. Illuminance (VIL) and circadian illuminance (CIL) [S3, S4]
The VIL is one of the yardsticks for measuring the brightness of light. The VIL means total amount of luminous flux per unit area from the incident light. As shown in Eq. S4, the CIL is calculated from the CAF and VIL. The CIL means total amount of circadian luminous flux per unit area from the incident light.
CIL (blm m 2 ⁄ , blx) = CAF (blm lm ⁄ ) × VIL (lm m 2 ⁄ , lx) Eq. S4 The visual illuminance (VIL) values were changed by the brightness, color, or CCT of light source. Therefore, the VIL of different light sources show different values even though the applied power of light source has the same input power. This means that if we use the VIL value as a yardstick for indicating brightness effect on human health, such as melatonin suppression value, we need to set the values of every light source. In contrast, the CIL value can be changed by only brightness of light source and the CIL shows almost same value regardless of light sources. Therefore, the CIL value is one of good yardsticks for indicating brightness effect on human health.

SI 2. Fabrication of monochromatic green, amber, and red phosphor-
converted LEDs. [S3, S5] To fabricate the monochromatic green(G), amber(A), and red(R) phosphor-converted LEDs (pc-LEDs), InGaN blue LEDs (λ max =445 nm) were used as an excitation source and phosphors (G: (Ba,Ca,Sr) n SiO 4 :Eu, A: (Ba,Ca,Sr) 3 SiO 5 :Eu, R: (Sr,Ca)AlSiN 3 :Eu). Phosphor paste, mixed with silicone binder and phosphors, were injected into B LED cup and hardened in an oven. After then, the long-wavelength pass dichroic filters (LPDFs) were capped on the G, A, and R pc-LEDs to realize monochromatic color by reflecting blue excitation source not to color mixing with excitation source and phosphor emission source. The fabrication methods for LPDFs were explained in our previous work. [S5] Figure S2 shows the emission SPD and 1931 CIE color coordinates for B LED, and LPDF-capped monochromatic G, A, and R pc-LEDs  CS flies were first exposed to LL, entrained to CH/CL cycles for 7 days, and then exposed to constant CL light. Heads of CS flies were collected on day 5 of CH/CL and day 2 of constant CL at indicated time points. Head extracts were obtained and analyzed by immunoblotting with anti-PER antibody. Actin was used as a loading control. ZT, zeitgeber time.